Oxygen gradient hydrogel drug screening

Abstract

The present invention describes methods for quantifying and analyzing cell migration and drug screening. Such methods include a gel (or a hydrogel) comprising a polymer, and cells that forms an oxygen gradient within the gel by controlling the balance of the diffusion of oxygen through the top of the gel and by the consumption of oxygen uptake by the cells. The migration of the cells is determined while the cells are grown in the gel of the present invention.

Claims

1. A method of analyzing cell migration comprising: providing a gel comprising a top, a polymer, and cells; forming an oxygen gradient within the gel by controlling a balance of the diffusion of oxygen through the top of the gel and of the consumption of oxygen uptake by the cells; and measuring speed, distance, or direction of cell motility or any combination thereof, thereby analyzing cell migration; wherein analyzing cell migration comprises immunofluorescent staining of the cells and a fluorescent microscope; wherein the step of analyzing cell migration quantifies the amount cell migration when comparing the cell migration of the cells with reference cell migration of reference cells in a reference sample.

2. The method of claim 1, wherein the oxygen gradient formed within the gel is in the range of about 0% to 21% of dissolved oxygen content.

3. The method of claim 1, wherein the gel further comprises a bottom and the cells are in the bottom ⅔ of the gel.

4. The method of claim 1, wherein the cells are cancer cells.

5. The method of claim 1, wherein the polymer is selected from the group comprising collagen, gelatin, chitosan, heparin, fibrinogen, hyaluronic acid, chondroitin sulfate, pullulan, xylan, dextran, and polyethylene glycol as well as their derivatives, and combinations thereof.

6. The method of claim 1, wherein the polymer is crosslinked by a cross linking agent selected from the group consisting of physical crosslinkers, chemical crosslinkers, enzyme mediated crosslinkers, peptide based crosslinkers, or a combination thereof.

7. The method of claim 6, wherein the cross linking agent is a phenolic agent selected from ferulic acid (FA), tyramine (TA), 4-Hydroxyphenylacetic acid, 3-(4-Hydroxyphenyl)propionic acid, Dopamine, Norepinephrine, epinephrine, and their derivatives.

8. The method of claim 1, wherein the cells deposit their own extracellular matrix in the gel.

9. The method of claim 1, wherein the oxygen gradient is determined by measuring the concentration of dissolved oxygen in at least two regions of the gel.

10. The method of claim 9, wherein the dissolved oxygen is measured using O.sub.2 sensors.

11. A method of identifying agents for treating or preventing cancer comprising: providing a gel comprising a top, a polymer, and cancer cells; forming an oxygen gradient by controlling a balance of the diffusion of oxygen through the top of the gel and by the consumption of oxygen uptake by the cancer cells; applying an agent to the gel; and comparing a characteristic of the cancer cells in the gel to cancer cells in a reference gel wherein the agent has not been applied to the reference gel, wherein the agent reduces or inhibits the characteristic of the cancer cells in the gel compared to the cancer cells in the reference gel, thereby identifying agents for treating or preventing cancer.

12. The method of claim 11, wherein the oxygen gradient formed is in the range of about 0% to 21% of dissolved oxygen content.

13. The method of claim 11, wherein the gel further comprises a bottom and the cancer cells are near the bottom of the gel.

14. The method of claim 11, wherein the polymer is selected from the group comprising collagen, gelatin, chitosan, heparin, fibrinogen, hyaluronic acid, chondroitin sulfate, pullulan, xylan, dextran, and polyethylene glycol as well as their derivatives.

15. The method of claim 11, wherein the polymer is crosslinked by a cross linking agent selected from the group consisting of physical crosslinkers, chemical crosslinkers, enzyme mediated cross linkers, peptide based crosslinkers, or a combination thereof.

16. The method of claim 15, wherein the cross linking agent is a phenolic agent selected from ferulic acid (FA), tyramine (TA), 4-Hydroxyphenylacetic acid, 3-(4 Hydroxyphenyl)propionic acid, Dopamine, Norepinephrine, epinephrine, and their derivatives.

17. The method of claim 11, wherein the cancer cells are selected from the group comprising sarcoma cells, carcinoma cells, or a combination thereof.

18. The method of claim 11, wherein the cancer cells are obtained from a tumor of an animal or a human subject.

19. The method of claim 11, wherein the cancer cells deposit their own extracellular matrix.

20. The method of claim 11, wherein the characteristic is selected from the group comprising gene expression, protein expression, cytoskeleton organization, nucleus shape, cell shape, ECM secretion and assembly, matrix degradation, matrix remodeling or a combination thereof.

21. The method of claim 11, wherein the characteristic is gene expression, wherein the mRNA expression is measured of a gene selected from the group comprising HIF-1a, ColA1, LOX, PLOD2, or a combination thereof.

22. The method of claim 11, wherein the characteristic is cell migration and the cell migration is determined by taking images of different regions within the hydrogels and using cell tracking to measure the distance from the edge of the tumor to the end of the tumor invading the gel.

23. The method of claim 22, wherein the cell tracking is determined by confocal z-stack analysis.

24. The method of claim 11, wherein the reference hydrogel is a non-hypoxic hydrogel having a dissolved oxygen content of greater than 5%.

25. The method of claim 11, wherein the gel has a thickness in the range of about 1.0 mm to 4.0 mm.

26. The method of claim 11, wherein the agents are selected from the group comprising a capture molecule, protein, peptide, nucleic acid, chemical, or a combination thereof.

27. The method of claim 11, wherein the agent is a capture molecule selected from the group comprising an antibody, an antibody fragment, an aptamer, a monoclonal antibody, or a combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A-1G illustrates enhanced invasion of sarcoma tumor grafts in hypoxic hydrogels. (A) In situ DO measurements in KIA tumors. (B) H&E stains and (C) HIF1-α stains (left) and quantification (right) of small and large tumors. Scale bars, 200 μm for the 4× images and 20 μm for the 40× images. (D) Schematic illustration of tumor encapsulation within HI hydrogel matrix. (E) DO levels of HI hydrogels encapsulated with tumor biopsy in hypoxic and nonhypoxic matrixes up to day 7 in culture (F) Left: Light microscope (left) and fluorescence microscope (right) images of sarcoma tumors encapsulated within nonhypoxic and hypoxic matrices (phalloidin in green; nuclei in blue). H, hydrogels; T, tumors. Scale bars, 100 μm. Right: Quantitative analysis of the sarcoma tumor invasion into hydrogel matrix, (G) Immunofluorescence staining and quantification of collagen deposition by tumor grafts cultured for 7 days (collagen in red; nuclei in blue). Scale bars, 25 μm. Significance levels were set at *P<0.05, **P<0.01 and ***P<0.001.

(2) FIG. 2A-2D illustrates hypoxia promote primary sarcoma migration. (A) Light microscope (left) and fluorescence microscope (right) images of day 3 images of KIA-GFP sarcoma tumors encapsulated within nonhypoxic and hypoxic matrices. Migrating GFP cells were tracked to determine (B) Three-dimensional trajectories of tracked cells (representative trajectories) (C) Overall speed, and (D) Mean square displacement (MSD) in the x, y, and z directions. Plots were created using position of KIA-GFP cells in the hydrogels. Significance levels were set at *P<0.05, {circumflex over ( )}P<0.01 and .sup.#P<0.001.

(3) FIG. 3A-3G illustrates Sarcoma cells remodel the hypoxic hydrogel. (A) Non-invasive DO readings at the bottom of the hypoxic and nonhypoxic hydrogel-sarcoma cell constructs. (B) Sarcoma cells encapsulated within HI hydrogels incorporating DQ™ gelatin for 3 days: (i) Representative fluorescence microscopy images. Scale bars, 25 μm and (i)) Quantitative analysis of relative fluorescence intensity (RFU). (C) Young's modulus (Pa) of the hypoxic and nonhypoxic hydrogels on day 0 and after 3 days of culture. (D) Real-time RT-PCR analysis of collagen modification genes. (E) Immunofluorescence staining and analysis of collagen deposition by the encapsulated cells (collagen in red; nuclei in blue). Scale bars, 50 μm. The effect of HIF1-α suppression in encapsulated sarcoma cells (KIA_scr, a control; KIA_HIF-1α(−), HIF knock down) after 7 days in culture was analyzed including: (F) collagen modification gene expression and (G) collagen deposition and quantification (collagen in red; nuclei in blue). Scale bars, 50 μm. Graphical results shown as the average value±s.d. Significance levels were set at *P<0.05, **P<0.01 and ***P<0.001.

(4) FIG. 4(A)-4(E) illustrates efficient sarcoma cell migration in hypoxic gradients (A) (i)—Illustration of hypoxic and nonhypoxic O.sub.2 gradient in HI hydrogels. (ii)—Invasive DO readings showing gradients in the HI hydrogels on Day 1 and 3. KIA-GFP cells were tracked on day 3 in hypoxic and nonhypoxic hydrogel to determine (B) Three-dimensional trajectories of tracked cells (representative trajectories), (C) Overall speed (D) Velocity in the x, y, and z directions; and (E) Mean square displacement (MSD) in the x, y, and z directions. Plots were created using position of KIA-GFP cells in the hydrogels. Significance levels were set at *P<0.05, {circumflex over ( )}P<0.01 and .sup.#P<0.001.

(5) FIG. 5(A)-5(G) illustrates minoxidil inhibit sarcoma cell migration and matrix remodeling in hypoxic hydrogel. KIA-GFP cells were tracked on day 3 in hypoxic treated and untreated hydrogel to determine (A) Three-dimensional trajectories of tracked cells (representative trajectories), (B) Overall speed (C) Velocity in the x, y, and z directions; and (D) Mean square displacement (MSD) in the x, y, and z directions. Plots were created using position of KIA-GFP cells in the hydrogels. (E) Collagen deposition and quantification (collagen in red; nuclei in blue). Scale bars, 50 μm. (F) Proteolytic degradation of HI hydrogels incorporating DQ™ gelatin for 3 days: (i) Representative fluorescence microscopy images. Scale bars, 20 μm and (ii) Quantitative analysis of relative fluorescence intensity (RFU). (G) Western blot analyses for PLOD2 in KIA cells cultured in hypoxic conditions with and without minoxidil treatment. Significance levels were set at *P<0.05, {circumflex over ( )}P<0.01 and .sup.#P<0.001.

(6) FIG. 6(A)-6(F) illustrates a hypoxic collagen gel. A.) Primary patient sarcoma collagen architecture, which was used to verify in-vitro collagen design. B.) Oxygen data showing the establishment of hypoxic and atmospheric gradients in the system. C.) Seeding technique used in the hypoxic collagen gel system. D.) Cells seeded in the hypoxic collagen gel matrix spreading out in the gel. E.) Cells moving in the hypoxic collagen gels at faster speeds in the z direction. F.) Drug screening data of camptothecin, CRXL101 (nanoparticle form of camptothecin) and DMSO control in collagen gels.

DETAILED DESCRIPTION OF THE INVENTION

(7) Intratumoral hypoxia occurs when the partial pressure of O.sub.2 falls below 5% and is a commonly observed feature of many sarcomas. Regional hypoxia develops as rapidly growing tumors outstrip their blood supply and as a consequence of aberrant tumor angiogenesis. As a result, O.sub.2 gradients develop throughout the growing tumor. Tumor hypoxia promotes chemo- and radiation resistance, primarily due to limited perfusion and reduced generation of reactive oxygen species (ROS), respectively. Moreover, the stabilization and activation of Hypoxia Inducible Factor (HIF) transcriptional regulators promotes adaptation to hypoxic stress by modulating tumor cell metabolism, survival, angiogenesis, migration, invasion, and metastasis. Elevated HIF expression has been associated with poor prognosis in many cancers and correlates with reduced survival for sarcoma patients. Recent transcriptome analyses have identified HIFs and HIF target genes as independent prognostic indicators of clinical outcomes. Finally, high levels of intratumoral hypoxia and HIF1-α accumulation are among the most important predictors of metastatic potential in patients with sarcoma, although the underlying mechanisms for this correlation remain incompletely characterized. Importantly, while the effect of overall reduced O.sub.2 on sarcoma cell responses has been studied; these cells are actually subjected to O.sub.2 gradients. Currently, the impact of specific oxygen gradients on sarcoma cell migration is unclear.

(8) The inventors found that deposition of immature collagen networks facilitated tumor cell metastasis to the lung in a HIF-1α dependent manner. The inventors also demonstrated that the HIF-1α regulated sarcoma metastasis through upregulation of procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD2) and the resulting increase in lysine hydroxylation of collagen molecules. The inventors have shown that PLOD2 expression promotes metastasis in a hypoxia- and HIF-1α dependent manner in a genetic in vivo model of UPS. However, the inventors do not yet know how sarcoma cell migration/invasion is altered in the presence of the O.sub.2 gradients that develop in tumors.

(9) Using in situ O.sub.2 measurements, the inventors found that hypoxia gradients exist in small primary mouse sarcoma tumors while large primary mouse sarcoma tumors contain severe hypoxic cores (≤0.1% pO.sub.2). To model intratumoral O.sub.2-gradients the inventors used novel O.sub.2-controlling hydrogels that can serve as 3D hypoxic microenvironments. In these hypoxia-inducible (HI) hydrogels, O.sub.2 is consumed while polymerization occurs resulting in spatial O.sub.2 gradients. Thus, with these hydrogels we can mimic physio-pathological O.sub.2 gradients. By encapsulating small tumor grafts in the hydrogels, we found that hypoxic gradients promoted cell invasion with faster speeds and longer distance, compared to nonhypoxic gradients. The inventors next demonstrate that the HI hydrogel culture system replicates HIF-1α-dependent collagen remodeling by sarcoma cells. Using this system, the inventors then showed that the hypoxic gradients guide the speed, distance, and direction of sarcoma cell motility compared with nonhypoxic hydrogels. Finally, the inventors showed that treatment of the encapsulated sarcoma cells with minoxidil abrogated cell migration and matrix remodeling in the O.sub.2 gradient.

(10) Primary Sarcoma Grafts Invade Hypoxic Hydrogels.

(11) To ascertain the physiological range of O.sub.2 gradients in the developing sarcoma tumor, we began by measuring dissolved O.sub.2 (DO) levels during the growth of primary mouse sarcoma tumors. The primary sarcoma tumors were generated in nude mice using murine sarcoma cells derived from Kras.sup.G12D/+; Ink4a/Arf.sup.fl/fl tumors (KIA). O.sub.2 gradient measurements during growth of subcutaneous primary sarcomas showed that in large tumors (>300 mm.sup.3) about 50% of the tumor mass is hypoxic (≤0.1% pO.sub.2). Smaller tumors exhibit hypoxic gradients throughout the tumor mass ranging from 0.1% pO.sub.2 at the center, to >6% pO.sub.2 in the outer layer bordering the edge of the tumor (FIG. 1A). Histological analysis further revealed the severe hypoxic tumor core with cells expressing HIF-1α localized to the nuclei in the larger tumor, while weaker and more diffused HIF-1α signal was observed throughout the entire smaller tumors (FIG. 1B-C). Previously, the inventors have established O.sub.2-controlling hydrogels and found that after hydrogel formation, the DO levels at the bottom of hydrogels decreased as gel thickness increased due to oxygen diffusion limitation, resulting in a broad range of O.sub.2 tensions within the gel matrices. Based on the oxygen gradient found in the xenograft tumors we sought to use the O.sub.2-controllable hydrogel system to provide a more physiologically relevant 3D microenvironment to study cell migration. Using the hypoxic hydrogel system the inventors recreated the hypoxic DO conditions found in the subcutaneous in vivo tumors and evaluated the role of O.sub.2 in 3D tumor cell migration assay. Tumor biopsy punches from smaller tumors were cut into 8 mm sections and grafted into the hypoxic and nonhypoxic hydrogels (FIG. 1D). Using noninvasive DO measurements at the bottom of the hydrogels, we found that DO levels in the hypoxic hydrogels reached <5% pO.sub.2 within initial 30 min and remained there during the entire week of measurements. Nonhypoxic hydrogels exhibited a higher level of O.sub.2 (>5% partial pressure of O.sub.2) during this culture period (FIG. 1E). The tumor engrafted within hypoxic matrix demonstrated increased invasion compared with tumors engrafted within nonhypoxic matrix. Specifically, tumors in hypoxic matrix invaded further into the hydrogel and away from the primary graft (after 1 week, 610±210 μm) compared to those encapsulated in nonhypoxic gels (after 1 week, 410±130 μm) (FIG. 1F). Moreover, migrating cells exiting tumor grafts in the hypoxic hydrogel deposited new collagen compared to nonhypoxic hydrogels (FIG. 1G).

(12) Cell Migration from Sarcoma Grafts is Regulated by O.sub.2 Gradients.

(13) To further investigate the effect of O.sub.2 gradients on sarcoma tumor migration, we performed real-time confocal analysis. Tumors generated from green fluorescent protein (GFP) positive KIA cells were engrafted in hypoxic and nonhypoxic hydrogels and imaged on day 3 when we first detected cell invasion into the hydrogel (FIG. 2A). We then analyzed cell migration on day 3, when we could first detect cell invasion from the tumor to the hydrogel. The inventors observed dynamic cell movement in the hypoxic constructs compared to the nonhypoxic constructs with more cells migrating out of the grafts under hypoxic conditions (FIG. 2B). Cell velocity analysis did not indicate specific directionality of migration with most cells moving in the x and y planes, suggesting a random migration path independent of O.sub.2 tension. However, we found a higher migration speed in hypoxic grafts compared to nonhypoxic grafts (FIG. 2C). Examining mean square displacement (MSD) in the three planes, we found that cells migrating in the hypoxic gradients move to larger distances compared to the nonhypoxic gradients with significantly longer distance in the z-direction (FIG. 2D) suggesting that while cells typically migrated in the x and y directions, those that migrated in the z-direction exhibited higher persistence. Overall, these data show that hypoxic gradient promotes tumor cell migration.

(14) Sarcoma Cells Remodel Collagen in Hypoxic Hydrogels.

(15) While the tumor graft model provides vital information, the inherited heterogeneity of the system limited the mechanistic insights that such system can provide. To more accurately model the effect of DO gradients on sarcoma cell invasion/migration within a complex tumor microenvironment, we next examined individual sarcoma cells embedded in the HI hydrogel. Non-invasive measurements of DO at the bottom of the hydrogel confirmed that the hypoxic matrix maintains low O.sub.2 levels during the 7 days of culture compared to nonhypoxic matrix (FIG. 3A). Notable, by day 7 in culture oxygen levels have decreased to ˜0.5%, likely to due to expansion of the cell number over the longer culture period and the associated cellular oxygen consumption. An important feature of this culture system is the ability to maintain the hypoxic gradient environment without exposure to high oxygen levels thus minimizing the introduction of reactive oxygen species (ROS). This, alongside with the ability to perform live-imaging of the cells while monitoring DO levels present a unique opportunity to link cellular responses to DO gradients. Growing evidence suggests that ECM remodeling is critical in sarcoma migration and metastasis. In particular, proteolytic degradation as well as abnormal collagen deposition and modification within hypoxic tumor microenvironments have been implicated as important parameters that enhance tumor invasion and metastasis. The gelatin-based HI hydrogels provide matrix adhesion and degradation sites similar to those in the tumor microenvironment, yet are collagen-free to prevent confounding results. We first tested whether sarcoma cells embedded in hypoxic hydrogel remodel the matrix material. The inventors examined the proteolytic degradation of the HI gelatin matrices using DQ™ gelatin that emits green fluorescence when degraded by a protease secreted by the cells. Interestingly, we observed higher fluorescence intensity in the cells cultured within the hypoxic microenvironments (480 RFU) compared to nonhypoxic matrix (50 RFU) (FIG. 3B). Rheological analysis further confirmed the softening of the hypoxic matrix (young's modulus of 20 Pa) compared to nonhypoxic matrix (young's modulus of 45 Pa) within 3 days of culture (FIG. 3C). The gelatin-based HI hydrogels further enabled us to examine whether sarcoma cells modify collagen in the hypoxic matrix. The inventors next examined the upstream effect of HIF-1α activation on relevant genes on days 3 and 7. While no change in gene expression was detected on day 3 of culture, we observed an upregulation of Col1A1, LOX, and PLOD2 expression after 7 days of culture in the hypoxic hydrogel compared to the nonhypoxic hydrogel (FIG. 3D). Moreover, at this time point, we detected collagen expression and deposition (FIG. 3E) as well as HIF-1α expression by the sarcoma cells in the hypoxic hydrogel. To determine whether ECM remodeling is regulated by the HIF-1α in the hypoxic matrix, we used short hairpin RNA (shRNA). The inventors detected significant down regulation of collagen modifying genes, Col1A1, LOX, and PLOD2 when HIF-1α expression was inhibited (FIG. 3F), resulting in less collagen deposition in hypoxic hydrogel encapsulated cells compared to controls (FIG. 3G). Overall, these results demonstrate that the 3D hypoxic hydrogel, regulates sarcoma matrix remodeling through hypoxic induction of HIF-1α expression. These results are consistent with various sarcoma studies in vivo, suggesting that the HI hydrogels are an appropriate 3D model with the necessary biological attributes to study sarcoma cell invasion and migration.

(16) O.sub.2 Gradients Modulate the Speed, Distance and Directional Bias of Sarcoma Cell Motility.

(17) The HI-hydrogel system is designed to create an O.sub.2 upward gradient, wherein DO levels increase toward the interface between the construct and O.sub.2 saturated culture media. Encapsulation of individual cell suspension would provide the inventors the opportunity to document single-cell movement in relation to the O.sub.2 gradient (FIG. 4Ai). As these constructs are cultured in air-oxygenated media, hypoxic and nonhypoxic upward gradients are maintained in each of the gel type. Invasive DO measurements validated hypoxic and non-hypoxic gradients in the hydrogel-cell constructs (FIG. 4Aii). These results confirm that the inventors are able to successfully mimic the gradients seen in the primary tumor in vivo. Thus, we next examined how sarcoma cell motility is regulated by the O.sub.2 gradients in the 3D hypoxic and nonhypoxic gradients. We encapsulated KIA-GFP in the HI hydrogels and analyzed movement on day 3 using real-time 3D cell tracking. Upon examining the 3D trajectory profiles of the KIA-GFP encapsulated cells, we observed greater overall cell movement in the hypoxic gradients compared to the nonhypoxic gels (FIG. 4B). We also found that cells in the hypoxic gradient gels are moving faster than those in the nonhypoxic gels. This holds true for the velocity profiles in the x, y, and z directions as well as the overall speed of the cells moving through the gel (FIG. 4C-D). Interestingly, we found that cells also moved in the z-direction, which has not been reported before. Importantly, cell velocity in the z-direction was primarily upwards, in the direction of increased O.sub.2 tension (FIG. 4D). We further computed and analyzed the MSD in the three planes. (FIG. 4E). Here too, O.sub.2 gradient seem to affect cell motility as indicated by the MSD in the z-direction. Cells exposed to the hypoxic gradients cells are traveling over larger distances compared to those in the nonhypoxic gradients (FIG. 4E). Interestingly, non-gradient hypoxic constructs (i.e. hydrogel constructs cultured in hypoxic conditions) showed slower cell movement and a lower mean squared displacement compared with hypoxic gradient constructs. Overall, this data shows that increased DO levels enhance the speed, distance and direction of sarcoma cell movement.

(18) Inhibiting 3D Hypoxic Gradient Migration.

(19) The inventors demonstrated that PLOD2 promotes metastasis in a hypoxia and HIF-1α dependent manner in an in vivo model of UPS. To examine whether the oxygen-controllable hydrogel faithfully represents the intratumoral hypoxic environment, we examined the effect of minoxidil, a pharmacologic inhibitor of PLOD2 expression, on sarcoma cell migration. Minoxidil treatment (0.5 mmol/L) for 12 hours of sarcoma cells encapsulated in the hypoxic hydrogels, significantly reduced KIA cell movement (FIG. 5A) concomitant with reduced overall cell speed as well as velocity and MSD in the x.y. and z directions (FIG. 5B-D). Minoxidil treatment of sarcoma cells encapsulated in the nonhypoxic hydrogels did not significantly affect cell migration in the X and Y directions, with slight inhibition of migration in the z-direction. Examining matrix remodeling, we found that minoxidil treatment reduced collagen deposition (FIG. 5E) and inhibited the proteolytic degradation of the hypoxic matrices (FIG. 5F). As expected, we found that hypoxia-induced PLOD2 expression is inhibited with Minoxidil treatment (FIG. 5G). Together, this data shows that hypoxia gradients determine the direction and speed of sarcoma cell migration in a HIF1/PLOD2 dependent manner. Importantly, these findings also highlight the utility of hydrogel encapsulation assays for the identification of novel therapeutic targets and inhibitors for the treatment of metastatic sarcoma and potentially other cancers as well.

(20) Leveraging our novel O.sub.2 controlling hydrogel, we generated a 3D in vitro model that enables us to analyze cancer cell responses to O.sub.2 gradients and the effect of small molecule inhibitors. Using this approach, the present invention presents a new concept in which O.sub.2 acts as a 3D physico-tactic agent during early stages of sarcoma tumor invasion. The inventors found that an O.sub.2 gradient is present in early stages of sarcoma development and that during this stage, cells respond to the hypoxic gradient by aggressively invading the matrix, followed by fast and long-distance migration. Moreover, the present invention demonstrated that in hypoxic gradients individual sarcoma cells not only migrate faster and over a longer distance while remodeling the matrix, they also migrate in the direction of increased O.sub.2 tension. Finally, we showed that treatment with minoxidil inhibits the migration and matrix remodeling in the hypoxic gradient. These findings are important for the understanding of the metastatic process and establishing the 3D in vitro model as a platform for testing therapeutic targets and interventions for the treatment of cancer.

(21) Oxygen-Controllable and Hypoxia-Inducible (HI) Hydrogels of the Present Invention.

(22) The oxygen Controllable and Hypoxia-Inducible Hydrogels are described in U.S. application Ser. No. 14/536,392, filed Nov. 7, 2014, and incorporated herein by reference. HI hydrogels can be generated with various phenolic agents (phenol molecules), such as ferulic acid (FA), tyramine (TA), 4-Hydroxyphenylacetic acid, 3-(4-Hydroxyphenyl)propionic acid, Dopamine, Norepinephrine, epinephrine, and their derivatives. Such phenolic agents include the structures in Table 1.

(23) TABLE-US-00001 TABLE 1 Phenolic Agents

(24) The novel HI hydrogels can be generated from natural or synthetic polymers as the polymer backbone. Examples of natural or synthetic polymers include collagen, gelatin, chitosan, heparin, fibrinogen, hyaluronic acid, chondroitin sulfate, pullulan, xylan, dextran, and polyethylene glycol as well as their derivatives. Gelatin (Gtn) is one preferred polymer backbone due to its cell-response properties, including cell adhesion and proteolytic degradability, which are critical in vascular morphogenesis (Hanjaya-Putra, D. et al., Blood, 2011; 118:804-815; Davis, G. E. et al., Circulation research, 2005; 97:1093-1107). Gtn provides relatively simple functionalization with for example, FA, for the formation of intramural hypoxia for both in vitro and in vivo vascular inductions. This has been further explored as an invasion assay by controlling the level of hypoxia on the gel in order to look at cancer vasculature sprouting (Shen, Yu-I., et al. “Hyaluronic acid hydrogel stiffness and oxygen tension affect cancer cell fate and endothelial sprouting.” Biomaterials science 2.5 (2014): 655-665). Dextran is a further preferred polymer backbone, used in conjunction with a hydrophilic linker such as polyethylene glycol (PEG) due to modifiability, bioactivity and hydrophilicity, as well as the similarity of the properties to those of various soft tissues. The high content of hydroxyl functional groups in the Dex molecule allows the Dex to be converted or modified easily with other molecules. A chain of Dex polymer includes three hydroxyl groups per repeat unit, which can allow for a high degree of substitution (DS) of target molecules (Jin, R. et al., Biomaterials 2007, 28, 2791). In addition, Dex has excellent water solubility that enables easy control of the precursor solutions. Some polymers may incorporate adhesion sites, such as Arg-Gly-Asp, and additional degradability features, such as MMP-sensitive peptides, depending on the application (Cuchiara, M. P. et al., Advanced functional materials, 2012; 22:4511-4518; Khetan, S. et al., Nature materials, 2013; 12:458-465; DeForest, C. A. et al., Nature materials, 2009; 8:659-664).

(25) Methods for Forming Oxygen Concentration Gradient Collagen Gel.

(26) In one embodiment, gradient collagen gels are prepared from commercially available rat tail collagen. A tissue culture dish is coated with polyethylenimine (PEI) and glutaraldehyde to increase adhesion to the gel. A combination of 1 M sodium hydroxide (NaOH), M199 10×, M199 1×, and collagen are mixed at a ratio of 1 part NaOH, to 3.15 parts M199 10×, to 68.0275 parts M199 1×, 27.0125 parts collagen (when the starting concentration of collagen is at 9.15 mg/mL). The solution is then incubated on ice as previously described. The collagen gel solution is then mixed with sarcoma cells at a concentration to achieve a final concentration of 2.5 mg/mL of collagen and 2 million cells/mL. The hypoxic gradient is achieved by manipulating the principles of Fick's first law:

(27) $J=D\frac{\partial c}{\partial z}+R$

(28) where J is the flux of oxygen through the gel, D is the diffusion coefficient of the gel, dc/dz is change in oxygen concentration per change in height, and R is the cell oxygen consumption rate. By controlling the vessel that the gel is placed in (i.e. polystyrene tissue culture well) we can minimize the diffusion into the hydrogel through the sides and bottom, and only have oxygen transport through the top of the gel. The diffusion of oxygen through the top of the gel is balanced by the consumption oxygen by the cells, allowing a hypoxic gradient to be maintained.

(29) Using Oxygen Concentration Gradient Collagen Gel to Study Cancer.

(30) Cancer metastasis is a poorly understood process that results in 90% of cancer-associated deaths. It has recently become apparent that the tumor microenvironment significantly impacts metastatic progression, through extracellular matrix (ECM) remodeling, stiffness remodeling, cell to cell/matrix interactions, and spatial/chemical gradients. Collagen hydrogels have been used as the traditional in-vitro model for cancer metastasis and growth for and for the study of tumor extra-cellular matrix interactions though other hydrogels maybe used such as hyaluronic acid hydrogels. These collagen gels exhibit typical collagen fibril morphology that is seen in patient tumors and in the standard in-vivo platform (see FIG. 1A). However, this model lacks one of the key principles that is observed in every tumor environment, hypoxia or low oxygen.

(31) A novel hydrogel platform was developed in the present invention to study cancer cell movement and migration. Firstly, collagen gels were generated and verified that by controlling the oxygen diffusion coefficient in the gel and the cell consumption rate, hypoxic gradient hydrogels can be established (see FIG. 1B). Next, cells were encapsulated to examine their behavior within the gel. The goal was to explore the collagen remodeling and cell migration within the platform (see FIG. 1C). In the confocal reflective image (see FIG. 1D), cells were seen sprouting along and interacting with these native collagen fibers in the in-vitro hypoxic collagen gel model of the present invention. Furthermore, it is confirmed that in the hypoxic gel there is a faster migration of the cells in the oxygen gradient direction (FIG. 1E).

(32) These hydrogels were then tested with novel therapeutics that are affected by hypoxia. One of the trends in cancer is to take drugs that have previously failed in the clinic and encapsulate them in nanoparticles to improve their delivery and efficacy. However, while improvement of nano-particled drug performances can be documented in vivo, results in vitro demonstrate equal or lesser efficacy. Camptothecin is a known cancer therapeutic that has failed in the clinic due to the ability of the drug to be inactive once it has been administered intravenously. The inventors have tested a novel compound CRLX101 of Cerulean Pharmaceuticals, which is a nanoparticle encapsulation of camptothecin. From this data (see FIG. 1F), it was demonstrated the higher efficacy of the nanoparticle compared to its small molecule counterpart in hypoxia. This is similar to the clinical data reported by Cerulean. From this data it was demonstrated the capability of this hypoxic collagen gel platform to better physiologically mimic the tumor microenvironment.

Examples/Methods

(33) The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The following Examples are offered by way of illustration and not by way of limitation

(34) Materials.

(35) Gelatin (Gtn, type A from porcine skin, less than 300 bloom), laccase (lyophilized powder from mushroom, ≥4.0 units/mg), 3-methoxy-4-hydroxycinnamic acid (ferulic acid, FA), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), dimethyl sulfoxide (DMSO), and deuterium oxide (D.sub.2O) were supplied from Sigma-Aldrich (Saint Louis, Mo.) and used as obtained without purification. Dialysis membrane (molecular cutoff=3500 Da) was purchased from Spectrum Laboratories (Rancho Dominguez, Calif.).

(36) Synthesis of Gtn-HI Hydrogels.

(37) Gelatin-based hypoxia-inducible (Gtn-HI) hydrogels were synthesized by carbodiimide-mediated coupling reaction as we previously reported. Prior to polymer synthesis, the inventors prepared a solvent by mixing DMSO and distilled (DI) water with 1 to 1 volume ratio. The inventors dissolved gelatin (1.0 g) in 50 ml of the solvent at 40° C. FA (0.78 g, 4.0 mmol) was dissolved in 20 ml of the solvent and activated with EDC (0.92 g, 4.8 mmol, 1.2 eq. of carboxyl unit of FA) and NHS (0.64 g, 5.6 mmol, 1.4 eq. of carboxyl unit of FA) at room temperature for 15 minutes to give amine reactive FA molecules. The activated solution was then applied to the Gtn solution and the conjugative chemical reaction was conducted at 40° C. After 24 hours, the reacted solution was dialyzed against DI water using a dialysis membrane (molecular cutoff=3500 Da) for five days. After dialysis, we obtained GtnFA polymers by freeze-drying and kept the product in a refrigerator (below 4° C.) before use. The chemical structure was confirmed using a .sup.1H NMR spectrometer (Bruker AMX-300 NMR spectrometer, Billerica, Mass.) and the degree of substitution of FA molecules was measured using an UV/Vis spectrometer (SpectraMax; Molecular Devices, Sunnyvale, Calif.).

(38) Cancer Cell Expansion and Culture within Gtn-HI Hydrogels.

(39) KIA (derived from a genetic murine model of sarcoma, LSL-Kras.sup.G12D/+; Ink4a/Arf.sup.fl/fl as established previously (2), or KIA-GFP or KIA-HIF-1α- or KIA-Scr were expended under standard culture conditions (37° C. and 5% CO.sub.2) in high-glucose DMEM with 10% fetal bovine serum (FBS), 1% Penicillin-Streptomycin (PS, Invitrogen) and 1% L-glutamine (Invitrogen). For cancer cell encapsulation, all solutions were prepared using Dulbecco's Phosphate-Buffered Saline (DPBS, Invitrogen) and filtered for sterilization using a syringe filter with a pore size of 0.2 μm. First, we prepared cell pellets of KIA or KIA-GFP or KIA-HIF-1α- or KIA-Scr (1.0×10.sup.6 cells) in 1.5 ml eppendorf tubes. The inventors then mixed the pellets with 375 μl of polymer stock solution (4% Gtn-FA solution) by gentle pipetting to give homogeneous cell suspensions and added 125 μl of laccase stock solution (100 U/ml). After mixing the enzyme, the solution was incubated at 37° C. for 2 minutes and then transferred to a 96-well plate (BD Bioscience). The cells were cultured within the hydrogel matrices under standard culture conditions in KIA media. The final concentration of cells, polymers, and laccase were 1.0-2.0×10.sup.6 cells/ml, 3%, and 25 U/ml, respectively. The cancer cell morphology was observed by light microscopy (in phase-contrast mode) and fluorescence microscopy (BX60, Olympus, Tokyo, Japan).

(40) Primary Tumor Formation, DO Measurements and Encapsulation.

(41) For primary tumor encapsulation, the inventors generated mouse sarcomas through subcutaneous injection of 1.0×10.sup.6 cells/ml of KIA into nude mice. After day 10 we isolated tumors and prepared tumor discs (diameter, 3.0 mm; thickness, 0.4 mm) by biopsy punching. The tumor specimens were encapsulated within the different thickness hydrogels (hypoxic, 2.5 mm, preferably 3 mm thickness; nonhypoxic, 1.25 mm thickness, preferably 1.5 mm). For tumor encapsulation, we first prepared 5 μl of the hydrogel pad (3 w/v % Gtn-HI) on a 96 well plate, and then placed the tumor specimens on the pad. The mixture of polymer and laccase solutions was applied to the wells, and entire hydrogel construct was cultured under standard cell culture conditions (37° C. and 5% CO.sub.2) in KIA media (high glucose DMEM with 10% FBS, 1% PS, and 1% L-glutamine) for up to a week. The O.sub.2 levels during the culture period were monitored using noninvasive O.sub.2 sensors as described above. For quantification of 3D tumor invasion, we took 3-5 images of different regions within the hydrogels using confocal z-stack analysis (>200 μm thickness) and measured the distance from the edge of tumor to the end of the tumor invading the hydrogel matrices using ImageJ software. To monitor real-time tumor invasion and migration, we encapsulated tumors generated from KIA-GFP cells.

(42) Non-Invasive O.sub.2 Measurement During Cell and Tumor Graft Culture.

(43) The DO levels were monitored non-invasively at the bottom of hydrogels using commercially available sensor patches (Presens, Regensburg, Germany), as previously established. For DO measurement, the cell suspension (75 μl of polymer and cell suspension) and laccase solution (25 μl of 100 U/ml laccase stock solution) were mixed and incubated for 2 min, and then plated on the O.sub.2 sensor attached to a 96-well plate (BD Bioscience). All measurements were performed under standard cell culture conditions (37° C. and 5% CO.sub.2) in the culture media. To vary O.sub.2 tension, we controlled the thickness of hydrogels in a volume-dependent manner. We generated hydrogels with different minimum dissolved O.sub.2 (DO.sub.min) levels (defined as hypoxic and nonhypoxic gels). For example, to generate hypoxic gels we plated 100 μl (2.5 mm thickness) of a mixture including polymer, cells, and laccase solutions into a well, whereas 50 μl (1.25 mm thickness) of the mixture was plated into the well for preparing nonhypoxic gels.

(44) Invasive O.sub.2 Gradient Measurements.

(45) The O.sub.2 levels in vivo were measured using Needle-Type Housing Fiber-Optic O.sub.2 Microsensor (PreSens, Regensburg, Germany). These needle sensors were mounted on a micromanipulator with 10 μm precision (PreSens). Hydrogel-cell constructs were generated as detailed above. Tumors were generated as previously stated, and DO measurements were performed once tumors were visible. O.sub.2 gradient measurements were performed using the needle sensor and a micromanipulator (PreSens). The tumor diameter was measured using a caliper and the needle was placed at the center of the tumor and moved outward in 0.5 mm increments, recording a DO reading at each distance, until reaching the edge of the tumor. The volume of the tumor was calculated using the following equation:
V=½*L*W.sup.2
where V is the volume of the tumor, L is the major axis, and W is the minor axis of the tumor. The tumor O.sub.2 measurements were averaged and the standard error mean was calculated for each distance from the center of the tumor.

(46) This same approach was used to evaluate the O.sub.2 gradient in the hydrogel at day 1, 3, 5 and 7 in the KIA encapsulated samples. It should be noted that in vitro invasive measurements might deviate a little from non-invasive measurements as the insertion of the needle can result in uncontrolled O.sub.2 penetration into the hydrogel.

(47) Matrix Degradation, Migration Assays and Drug Treatment.

(48) To assess effect of O.sub.2 levels on matrix degradation, we incorporated 10 μg/ml of DQ™ gelatin (Invitrogen) into polymer solutions when preparing cell suspensions, and then mixed with the laccase solution as described above. After day 3, the hydrogels with DQ-gelatin were observed by the fluorescence microscopy (BX60, Olympus, Tokyo, Japan) and quantified by measuring the fluorescence intensity using a fluorescence spectrophotometer at wavelength of 495 nm excitation and 515 nm emission (Molecular Devices).

(49) For the 3D cancer cell migration assay, we encapsulated KIA-GFP cells and tumor grafts in hydrogel to generate constructs with different O.sub.2 levels as previously. For non-gradient hydrogel controls, constructs were formed and incubated at 1% oxygen for 3 days and then tracked in that chamber at day 3. Cells were tracked at day 3 using live-cell three-dimensional confocal microscopy (LSM 780, Carl Zeiss, Thornwood, N.Y., USA) equipped with a cell incubator (5% CO.sub.2 and 37° C.). In order to properly optimize the experiment only cells that started in frame were included, with a shorter timeframe was used for the tumor encapsulated samples. The time-lapse and z-stack images (>200 μm thickness) were collected every 30 minutes up to 24 hours at five randomly selected positions. The images were analyzed using Imaris spot analysis (Imaris 8.1, Bitplane, South Windsor, Conn., USA) software to track the time-dependent mobility. The 3D migration analysis was performed following the strategy developed by Wirtz and colleagues. A minimum of 100 individual cells at each point were tracked to generate x, y, and z coordinates at each time point. This data was then sorted to only include cells that were present at time zero. From this sorted data the time that the cells were in frame was calculated, and the most common time was used to pick cells for tracking analysis. This was done to maximize the sample size of cells that could be analyzed. Finally, velocity and speed profiles, mean squared displacements, and trajectory plots were calculated using code adapted from Wirtz et al (5, 6) for triplicate tracking trials (n=3) (Matlab, Mathworks Inc.). The statistical analysis was performed using MATLAB (Mathworks Inc.) to calculate the mean, standard deviation and standard error mean. A t-test was performed where appropriate to determine significance (GraphPad Prism 4.02). Graphed data is presented as average±SD. Significance levels were set at: *P<0.05; {circumflex over ( )}P<0.01; .sup.#P<0.001.

(50) For minoxidil treatment, the cells were cultured in hypoxic hydrogels as stated above for three days. On the third day, 0.5 mM minoxidil (dissolved in KIA cell culture media) was added to the wells and the cells were tracked for 24 hours. Untreated cultures served as controls. Cell tracking and data analysis was performed as above.

(51) PLOD2 Western Blot.

(52) Low-oxygen conditions were maintained in a Ruskinn in vivO.sub.2 400 hypoxia work station. Simultaneously, KIA Cells were treated with vehicle or 0.5 mM Minoxidil diluted in DMEM culture media (Sigma Aldrich) for 16 hours. Whole cell lysates were prepared in SDS/Tris pH 7.6 lysis buffer. Proteins were electrophoresed and separated by SDS-PAGE and transferred to nitrocellulose membranes and probed with the following antibodies: rabbit anti-GAPDH (Cell Signaling Inc.), and rabbit anti-PLOD2 (Proteintech).

(53) Rheological Analysis.

(54) To analyze matrix stiffness, the inventors performed rheological analysis of the HI hydrogels using a rheometric fluid spectrometer (RFS3, TA Instruments, New Castle, Del.). In the rheological experiments, tumor constructs cultured within HI hydrogels were plated in the instrument. The inventors performed dynamic time sweep on the samples after day 3 in culture. The inventors monitored the elastic modulus (G′) and viscous modulus (G″) at 10 percent of strain and a frequency of 0.1 Hz at 37° C. A solvent trap wetted with deionized water was used to prevent sample evaporation.

(55) Gene Expression.

(56) To assess gene expression, we performed quantitative real time RT-PCR as described previously. Total RNA was extracted from cancer cells encapsulated in hydrogels using TRIzol (Invitrogen), according to the manufacturer's instructions. In brief, the inventors placed the hydrogel constructs into 500 μl of TRIzol and homogenized using a micro homogenizer. The suspension was centrifuged at 12,000 G for 15 minutes and the supernatant was separated. The inventors then added 100 μl of chloroform to the solution and mixed manually for 20 seconds. The mixture was centrifuged at 12,000 G for 10 minutes and the supernatant was isolated. The solution was mixed with 250 μl isopropyl alcohol and kept at −4° C. for 1 hour. The precipitates were separated by centrifugation at 7,500 G for 5 minutes and then washed using 70% ethyl alcohol (EtOH). Total RNA was quantified using an ultraviolet (UV) spectrometer and validated by lack of DNA contamination. One microgram of RNA was transcribed using reverse transcriptase M-MLV and oligo(dT) primers (both from Promega, Madison, Wis.), according to the manufacturer's instructions. The inventors used TaqMan Universal PCR MasterMix and Gene Expression Assay (Applied Biosystems, Foster City, Calif.), according to the manufacturer's instructions for LOX, PLOD2, col1A1, and β-actin.

(57) Histological Analysis.

(58) For histological analysis, hydrogel constructs or tumors were harvested and fixed using 3.7% paraformaldehyde and then dehydrated in graded EtOH (80-100%). We then embedded the samples in paraffin blocks and serially sectioned them using a microtome (5 μm). The slides were stained with either haematoxylin or eosin (H&E) or underwent immunohistochemistry for HIF-1α as previously described. For quantification of HIF-1α positive cells in tumor sections, cells expressing nuclear HIF-1α were counted manually from 40× images (n=8). The total number of cells in the images were counted and percent positive cells were calculated. The standard deviation and t-test were performed on the data to determine significance (GraphPad Prism 4.02). Graphed data is presented as average±SD. Significance levels were set at: P<0.05; **P<0.01; ***P<0.001.

(59) Immunoflorescence Analysis.

(60) Hydrogels were fixed using 3.7% paraformaldehyde and incubated at room temperature for 1 hour. For staining the cells were permabolized with 0.05% Triton-X for 30 minutes, washed with PBS and incubated with primary antibodies over night. The primary antibody used with collagen 1 or HIF-1α (Novous Biologicals, Littleton, Colo.). Certain samples were rinsed with PBS and incubated with Alexa Fluor 546 (1:500; Invitrogen) for 1 hour, followed by incubation with Alexa Fluor 488 Phalloidin (1:40; Invitrogen) for 30 minutes and with 4-6-diamidino-2-phenylindole, DAPI, (1:1000; Roche Diagnostics) for 10 minutes. The labeled cells were examined using fluorescence microscopy (LSM 780, Carl Zeiss, Thornwood, N.Y., USA). Collagen fluorescence intensity was analyzed using ImageJ (ImageJ, National Institutes of Health, Bethesda, Md.).

(61) Statistical Analysis.

(62) All experiments were performed in triplicate for at least 3 biological replicates. We performed RT-PCR analysis in triplicate with duplicate readings. We performed statistical analysis using GraphPad Prism 4.02 (GraphPad Software Inc., La Jolla, Calif.). For all analyses other than the live migration (which its statistics is detailed above), the standard deviation and t-test were performed on the data to determine significance (GraphPad Prism 4.02). Graphed data is presented as average±SD. Significance levels were set at: P<0.05; **P<0.01; ***P<0.001. All graphical data were reported.

(63) Gradient Collagen Gels.

(64) In one embodiment, gradient collagen gels are prepared from commercially available rat tail collagen. A tissue culture dish is coated with polyethylenimine (PEI) and glutaraldehyde to increase adhesion to the gel. A combination of 1 M sodium hydroxide (NaOH), M199 10×, M199 1×, and collagen are mixed at a ratio of 1 part NaOH, to 3.15 parts M199 10×, to 68.0275 parts M199 1×, 27.0125 parts collagen (when the starting concentration of collagen is at 9.15 mg/mL). The solution is then incubated on ice as previously described. The collagen gel solution is then mixed with sarcoma cells at a concentration to achieve a final concentration of 2.5 mg/mL of collagen and 2 million cells/mL.

(65) All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

(66) The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

(67) Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.