METHODS AND PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF OVARIAN CANCER, BREAST CANCER OR PANCREATIC CANCER
20220363776 · 2022-11-17
Inventors
Cpc classification
C07K16/2896
CHEMISTRY; METALLURGY
C07K2319/55
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
Experimental and clinical evidence suggests tumor-associated macrophages (TAM) play important roles in cancer progression. Here, the inventors show that the omentum is a critical pre-metastatic niche for development of invasive disease in this model and defined a unique subset of CD163+ Tim4+ tissue-resident macrophages in omentum of embryonic origin and maintained independently of bone marrow-derived monocytes. Transcriptomic analysis showed that resident CD163+ Tim4+ omental macrophages were phenotypically distinct and maintained their resident identity during tumor growth. Selective depletion of CD163+ Tim4+ macrophages in omentum using genetic and therapeutic tools prevented tumor progression and metastatic spread of disease. The molecular pathways of cross-talk between tissue-resident macrophages and disseminated cancer cells may represent new targets to prevent metastasis and disease recurrence. Thus the present invention relates to a method of treating ovarian cancer, breast cancer and pancreatic cancer in a subject in need thereof comprising administering to the subject a therapeutically effective of an agent capable of depleting the population of CD163+ Tim4+ tumor associated macrophages.
Claims
1. A method of treating a cancer or preventing resistance to an immune checkpoint therapy, radiotherapy or chemotherapy of the cancer in a subject in need thereof; the method comprising administering to the subject a therapeutically effective amount of an agent capable of depleting the population of CD163+ Tim4+ macrophages and CD163− Tim4+ macrophages in the subject's tumor; wherein the agent is an antibody having binding affinity for Tim4.
2-22. (canceled)
23. The method of claim 1, wherein the cancer is selected from the group consisting of ovarian cancer, pancreatic cancer and breast cancer.
24. The method of claim 1, wherein the cancer is ovarian cancer.
25. The method of claim 1, wherein the cancer is resistant to immune checkpoint therapy, radiotherapy or chemotherapy.
26. The method of claim 1, wherein the antibody binds to the extracellular domain of Tim4.
27. The method of claim 1, wherein the antibody is an antibody-drug conjugate.
28. The method of claim 1, wherein the antibody is conjugated to a cytotoxic moiety.
29. The antibody of claim 1, wherein the antibody is conjugated to doxorubicin and/or duocarmycin.
30. The method of claim 1, wherein the antibody mediates antibody-dependent cell-mediated cytotoxicity.
31. The method according to claim 1, further comprising administering to the subject a therapeutically effective amount of a second agent capable of depleting the populations of CD163+ Tim4+ macrophages and CD163+ Tim4− macrophages in the subject's tumor; wherein the second agent is an antibody having binding affinity for CD163.
32. The method according to claim 1, further comprising administering to the subject a therapeutically effective amount of a second agent capable of depleting the population of CD163+ Tim4+ and CD163+ Tim4− macrophages in the subject's tumor; wherein the second agent is an antibody having binding affinity for CD163; the method thereby leading to depletion of CD163+ Tim4+ macrophages, CD163− Tim4+ macrophages and CD163+ Tim4− macrophages in the subject's tumor.
33. The method of claim 1, wherein the antibody is a multispecific antibody comprising a first antigen binding site directed against Tim4 and a second antigen binding site directed against CD163.
34. The method of claim 1, wherein the antibody is a multispecific antibody comprising a first antigen binding site directed against Tim4 and a second antigen binding site directed against CD163; wherein the method is for depletion of CD163+ Tim4+ macrophages, CD163− Tim4+ macrophages and CD163+ Tim4− macrophages in the subject's tumor.
35. A method of treating a cancer or preventing resistance to immune checkpoint therapy, radiotherapy or chemotherapy of a cancer in a subject in need thereof; the method comprising administering to the subject a combination comprising an antibody having binding affinity for Tim4 and an antibody having binding affinity for CD163.
36. The method of claim 35, wherein the cancer is selected from the group consisting of ovarian cancer, pancreatic cancer and breast cancer.
37. The method according to claim 35, wherein the method leads to the depletion of CD163+ Tim4+ macrophages, CD163− Tim4+ macrophages and CD163+ Tim4− macrophages in the subject's tumor.
38. The method according to claim 34, wherein the antibody having binding affinity for CD163 and/or the antibody having binding affinity for Tim4 is an antibody-drug conjugate.
39. A kit of parts comprising: a first container comprising an antibody having binding affinity for Tim4; and a second container comprising an antibody having binding affinity for CD163.
40. The kit of parts according to claim 39, wherein the antibody having binding affinity for CD163 and antibody having binding affinity for Tim4 are antibody-drug conjugates.
41. The kit according to claim 39, wherein the antibody having binding affinity for CD163 and/or the antibody having binding affinity for Tim4 is conjugated to doxorubicin and/or duocarmycin.
Description
FIGURES
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EXAMPLES
Example 1—Material & Methods
[0142] Mouse Breeding and Ovarian Cancer Model
[0143] Tg(Csflr-LSL-HBEGFmCherry) (Schreiber et al., 2013), Rosa26.sup.LSL-YFP, Cx3Crl.sup.CreER and Cx3Clr.sup.GFP/+ were obtained from the Jackson Laboratory (Bar Harbor, Me., US). C57BL/6J mice were obtained from Janvier Labs (Saint-Berthevin, FR). Ccr2.sup.−/− mice and Rosa26.sup.LS-tdRFP mice were gifts from Bernard Malissen (Centre d'Immunologie Marseille Luminy, Marseille, France). Cd163.sup.iCre mice were generated from modified ES cells on a C57BL/6 background as described (Etzerodt et al, in press). In brief; a FlpO-NeoR cassette encoding IRES-iCre was inserted in the 3′UTR of the CD163 gene using homologous recombination and used to generate chimeric mice that were subsequently crossed to Flp deleter mice to facilitate removal of NeoR cassette. To mimick peritoneal spread of epithelial ovarian cancer, 1×10.sup.6 ID8-Luc cells were injected i.p in 500 μl sterile PBS pH 7.4. Tumor burden was estimated weekly by injecting mice i.p. with 100 mg/kg d-Luciferin followed by in vivo bioluminescence imaging using an IVIS Spectrum imager (PerkinElmer). For visualizing infiltration of ID8-cells in omentum, ID8 EOC cells were prelabeled with Qtracker 705 cell labeling kit (Thermo Fisher) prior to i.p. injections in accordance with manufactures instructions. For therapeutic treatment with lipid nanoparticles (LNP) mice were injected with 100 μl of LNP (1 mg/kg dxr) by retroorbital injection starting from 5 weeks after i.p injection of 1×10.sup.6. For prophylactic treatment with LNP or diphteria toxin, mice were injected i.p with either 200 μl LNP (1 mg/kg dxr) or 200 μl diptheria toxin (4 ng/kg) starting from 6 days prior to inoculation of tumor cells. All mice were euthanized at the indicated times and peritoneal lavages and tissues were collected for cytometric analysis or imaging analysis. Briefly, 3 ml of ice-cold PBS with 2 mM EDTA, pH 8.0 was injected intraperitoneally and after a careful massage to detach all the cells in the cavity, peritoneal fluid was collected through a 23G syringe. Tubes were weighed to determine the recovered lavage volume and the cell density was assessed using a hemocytometer. All mice were housed at the animal facility at Centre d'immunologie Marseille-Luminy with water and food ad libitum and 12h night/daylight cycle. All animal experiments were approved and carried out in accordance with the limiting principles for using animal in testing (the three R's, replacement, reduction and refinement) and approved by the French Ministry of Higher Education and Research.
[0144] Fate-Mapping Experiments
[0145] Genetic fate-mapping using Cx3Crl.sup.CreER:R26-YFP mice was performed as previously described (Mossadegh-Keller et al., 2017); pregnant females were pulse-labeled at E16.5 by intraperitoneal injection of 0.1 mg/kg tamoxifen and 0.05 mg/kg progesterone. For fate-mapping in adult tumor bearing mice, Cx3Crl.sup.CreER:R26-tdRFP mice were injected with 1×10.sup.6 ID8-luc cells i.p. and after 6 weeks pulse-labeled with a single dose of 2 mg tamoxifen dissolved in 200 μl corn oil by oral gavage. Generation of shielded chimeras was performed as previously described (Goossens et al., 2019); CD45.1 congenic mice were anaesthetized with Ketamine (150 mg/kg) and Xylazine (10 mg/kg) and placed in 6 mm thick lead cylinders, exposing only the hind legs. With the abdominal area protected, mice were irradiated with 9 Gy and reconstituted with 10.sup.7 bone marrow cells from CD45.1/.2 mice. After 5 weeks, chimerism of blood leukocytes was assessed by flow cytometry.
[0146] Flow Cytometry and Cell Sorting
[0147] Single cell suspensions were prepared from omentum by digesting the tissue in RPMI 1640 medium with 1 mg/ml Collagenase II (Sigma Aldrich), 50 μg/ml DNAseI (Roche, Hvidovre, DK) and 0.1% (w/v) BSA for 30 min at 37° C. with gentle agitation. Cell suspensions were subsequently passed through 70 μm cell strainer (BD Biosciences, FR) and collected by centrifugation. Blood and ascitic cells harvested by peritoneal lavage were used without further processing. For red blood cell (RBC) lysis, cell suspensions were incubated with 0.85% NH.sub.4Cl for 2 min at RT, collected by centrifugation and resuspended directly in FACS buffer (1×PBS pH 7.4, 1 mM EDTA pH 8.0, 3% FCS and 0.1% NaN.sub.3). For flow cytometry, single-cell suspensions were incubated at 4° C. for 10 min with 2.4G2 antibody for Fc receptor blocking followed by incubation with the specified antibodies (see Table 1 for details) for 30 min at 4° C. Prior to analysis, cells were incubated with Sytox Blue (Thermo Fischer Scientific, FR) to discriminate dead cells and filtered through a 70 μm cell strainer. ALDH activity in tumor cells was measured using the ALDEFLUOR Kit (Stem cell Technologies), in accordance with manufactures instructions. In brief, 2×10.sup.6 cells were incubated with 5 μl ALDEFLUOR stock solution or 5 μl ALDEFLUOR stock solution in combination with 5 μl of the ALDH inhibitor DEAB and incubated for 30 min at 37° C. ALDH activity was subsequently measured by flow cytometry relative to DEAB treated control cells. All flow cytometry analysis was performed with either BD FACS LSR-2 or Fortessa X-20 flow cytometers whereas cell sorting was performed with BD FACS Aria SORP. All cytometers were equipped with a 350 nm laser (BD Biosciences). Subsequent data analysis was performed using FlowJo software V10.4 for Mac (Tree Star).
[0148] Liposome Preparation
[0149] Long-circulating liposomes encapsulating doxorubicin were prepared and modified for CD163 targeting as previously described (Etzerodt et al., 2012)(Fritze et al., 2006). Briefly, liposome formulations were formed using the ethanol-injection method from a mixture of HSPC, mPEG2000-PE and Cholesterol (molar ratio of 55:40:5) (Lipoid GmBH, Ludwigshafen, Germany and Sigma Aldrich). Lipids were dissolved in EtOH at 65° C. for 15 min followed by hydration (to 10% EtOH) for 1 h at 65° C. in aqueous buffer suitable for further downstream applications. Liposomes were sized by extrusion 25 times through a 0.1 μm filter using the Avanti mini-extruder kit (Avanti Polar Lipids, AL, US) and dialyzed twice against 150 mM NaCl (0.9% NaCl) with second dialysis being over night at 4° C. For remote loading of doxorubicin, lipid was hydrated in 300 mM (NH.sub.4).sub.1HPO.sub.3. Following extrusion and dialysis, (NH.sub.4).sub.1HPO.sub.3 containing liposomes were mixed with doxorubicin-HCl for 30 min at 65° C. at a doxorubicin:lipid ratio at 1:5. Lipid content, drug content and encapsulation efficiency was subsequently estimated from high-pressure size-exclusion chromatography (UV absorbance 210 nm) using a Dionex Ultimate3000 HPLC system (Thermo Fischer Scientific, Hvidovre, Denmark) equipped with Ascentis C18 column (Sigma Aldrich). Liposome size was estimated using dynamic light scattering and the DynaPro NanoStar system (Wyatt Technology Europe GmbH, Dernbach, Germany). Modification of liposomes for CD163 targeting was done as described previously using the post-insertion method of αCD163 antibody, clone 3E10B10 (Etzerodt et al., 2012; Torchilin et al., 2001).
[0150] Immunohistochemistry and Whole-Mount Immunofluorescence Imaging
[0151] Omentum was fixed in 1% formalin and either embedded in OCT for cryostat sectioning or used directly for whole mount imaging. For histological analysis, 10 μm cryostat sections were mounted on glass slides and stained with hematoxylin and eosin and visualized on an upright microscope equipped with a 10× objective. For whole mount imaging, omental tissue was incubated with with anti-CD163-ATT0565 (Etzerodt et al., 2013), anti-CD169-eFluor660 (Clone Ser4; eBioscience), anti-CD45.2-Alexa488 (Clone 104; Biolegend) and Tim4-Alexa647 (RMT54-4; Biolegend) in 0.1M Tris pH 7.2, 1% Triton X-100, 0.5% BSA overnight at 4° C. Nuclei were visualized with Hoechst 33342 (Sigma Aldrich). Tissue was subsequently mounted in RapiClear 1.47 for tissue clearing on glass slides with 0.2 mm iSpacer (SunJin Lab Co. Hsinchu City, Taiwan). Images were acquired on a Zeiss LSM780 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, DE) using spectral unmixing with a 10× or 20× objective.
[0152] Spheroid Formation Assay
[0153] Tumor cells in ascites were enriched by depleting leukocytes in peritoneal lavage using the CD45.2 magnisort kit (eBioscience) and seeded at 40,000 cells per well in ultra-low attachment 96 well plates (Corning Life Science, UK) in DMEM supplemented with 4% heat-inactivated FCS. Formation of spheroids was subsequently monitored by microscopy using an inverted microscope equipped with a 4× objective.
[0154] RNA Sequencing and Bioinformatics Analysis
[0155] Library preparation and RNA sequencing (RNAseq) was performed by the GenomEast platform at Institut de Génétique et Biologie Moléculaire et Cellulaire, Strasbourg, France. Full length cDNA was generated using Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio Europe, Saint Germain en Laye, France) according to manufacturer's instructions from 500 cells isolated by cell sorting in PBS buffer containing RNAses inhibitor. cDNA was amplified with 14 cycles of PCR for cDNA amplification by Seq-Amp polymerase. Six hundred pg of pre-amplified cDNA were then used as input for Tn5 transposon tagmentation by the Nextera XT DNA Library Preparation Kit (96 samples) (Illumina, San Diego, Calif.) followed by 12 cycles of library amplification. Following purification with Agencourt AMPure XP beads (Beckman-Coulter, Villepinte, France), the size and concentration of library DNA were assessed on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina HiSeq4000 platform generating 50 bp reads. Samples were trimmed to remove TruSeq adapters using BBduk (Bushnell, 2014) and subsequently mapped to the mouse genome assembly version mm10 using STAR version 2.5.3a (Dobin et al., 2012) with junction annotation from Ensembl version 79 (Yates et al., 2016). Gene counts obtained directly from STAR were used in gene expression analysis with DESeq2 (Love et al., 2014) using cqn (Hansen et al., 2012) derived normalization factors. Self-organizing maps (SOM) clustering analysis was performed using the “kohonen” R pacakage (Wehrens and Buydens, 2007; Wehrens and Kruisselbrink, 2018), while comparative gene ontology analysis was carried out using clusterProfiler (Yu et al., 2012). Heatmaps and hierarchical clustering was generated using the One minus Pearson correlation and PCA plots with network analysis (using Pearson correlation) to show the 3 nearest neighbors were generated using Qlucore Omics Explorer (Qlucore AB, Lund, SE).
[0156] Gene Expression Analysis
[0157] Total RNA was purified from sorted cell populations using the RNeasy Micro Kit (Qiagen, Hilden, DE) and concentration determined using the Quant-IT RiboGreen RNA assay kit (Thermo Fischer Scientific). First strand cDNA synthesis was performed with High Capacity cDNA Reverse Transcriptase Kit (Thermo Fischer Scientific) followed by preamplification of genes of interest using the Fluidigm PreAmp Master Mix (Fluidigm Europe B.V. Amsterdam, NL) with 25 ng of total RNA and in accordance with the manufacturer's instructions. Exon-spanning primers to amply genes of interest were designed using Primer-Blast (see Table 2 for details). To increase sensitivity, genes of interest were pre-amplified by 12 cycles of PCR using pooled assays followed by exonuclease I treatment (New England Biolabs, MA, USA) to remove unincorporated primers. Final pre-amplified cDNA was diluted 1:5 in TE buffer. Gene expression analysis was carried out using the Biomark HD system from Fluidigm (Fluidigm Europe B.V.) in accordance with manufacturer's instructions and standard settings. Data was analyzed using the Real-Time PCR Analysis Software (Fluidigm Europe B.V.) and resulting CT values were normalized to Ppia to obtain dCT values.
[0158] Statistical Analysis
[0159] For treatment studies statistical analysis was performed using two-way ANOVA followed by Tukey post-hoc test. For comparison between groups, statistical testing was performed using non-parametric tests such as Mann-Whitney or Kruskal-Wallis followed by Dunn's multiple comparisons test. p values are indicated as *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All statistical analysis was performed with Graphpad Prism 7 for Mac.
Results
Example 2—the Omentum is a Critical Pre-Metastatic Niche for Ovarian Cancer Cells
[0160] The omentum is an adipose tissue formed from a fold of the peritoneal mesothelium. In humans the greater omentum covers the majority of the abdomen, whereas in mouse the omentum is only a thin stretch of adipose tissue located between the stomach, pancreas and spleen. The peritoneal spread of ovarian cancer can be modelled using the immortalized mouse ovarian epithelial cell line ID8 (Roby et al., 2000). The intra-peritoneal (i.p.) injection of ID8 cells leads to the development of diffuse peritoneal carcinomatosis and malignant ascites with a long latency period (up to 12 weeks). We used an ID8 cell line transduced to express firefly luciferase (ID8-luc) (Hagemann et al., 2008) to monitor tumor progression after i.p. injection by non-invasive bioluminescent imaging. We observed that ID8 cells localized primarily to the omentum for up to 35 days before spreading throughout the peritoneal cavity (data not shown). Infiltration of tumor cells into the omentum was confirmed by histological analysis, where visible tumor nodules could be observed within the adipose tissue from 35 days (data not shown). From day 63, the entire omentum was invaded by tumor cells and only residual adipose tissue remained (data not shown). Interestingly, despite the presence of obvious tumor nodules in omentum from day 35, accumulation of ascitic ID8 cells was not detectable until more than 50 days after i.p. injection (data not shown). Ultimately, ascites formation was associated the development of multiple distant metastases in the diaphragm and peritoneal wall (data not shown), reflecting late-stage disease in HGSOC patients. To establish the contribution of the omentum to the disease course in this model, we transplanted ID8-luc cells into omentectomized mice and monitored tumor growth. Whereas sham-operated and control mice showed a comparable course of tumor progression with accumulation of ascitic tumor cells after 63 days, we observed only minimal tumor growth in omentectomized mice and ascitic cells were barely detectable (data not shown).
Conclusion
[0161] These data suggested the omentum was a critical pre-metastatic niche for tumor progression in this model and not merely a receptive site for peritoneal metastasis.
Example 3—Ovarian Cancer Cells Colonize Fat-Associated Lymphoid Clusters in Close Contact with Omental Macrophages
[0162] The omentum has a particularly high density of fat-associated lymphoid clusters (FALC) that are thought to be important structures for capturing peritoneal antigens (Rangel-Moreno et al., 2009). Previous studies have proposed that FALC promote the colonization of omentum by ovarian cancer cells, however, neither B or T lymphocytes were shown to contribute to tumor growth (Clark et al., 2013). To visualize the localization of ID8 cells in the omentum, we labeled cells with Qdots (Qtracker®705) before i.p. injection and analyzed omentum by whole-mount confocal imaging. One day after injection, we observed that ID8 cells were located in the vicinity of FALCs, an area densely populated by omental macrophages (data not shown). To further characterize omental macrophages, we analyzed omentum by flow cytometry. Gating on the CD11b+ myeloid cell fraction (CD45.2+ Linneg (CDS, CD19, NK1.1, Ly6G, SiglecF) CD11b+) we identified macrophages as F4/80+CD64+ cells, whereas monocytes were F4/80− CD64− CCR2+(data not shown). To further characterize the heterogeneity of omental macrophages we analyzed expression of these markers on F4/80+ CD64+ cells. We observed a gradient of CD169 expression by omentum macrophages, where only CD169hi cells co-expressed Lyve1 (data not shown). Further analysis showed that CD169-Lyve1− and CD169int Lyve1− cells were also negative for CD163 and Tim4 expression (data not shown), indicating a less mature phenotype. In contrast, CD169hi Lyve1+ cells could be separated into four distinct populations based on CD163 and Tim4 expression; CD163+ Tim4+ (P1), CD163+ Tim4− (P2), CD163− Tim4− (P3) and CD163− Tim4+ (P4) (data not shown). Next, we analyzed the localization of these macrophage subsets in omentum by whole-mount confocal imaging. We found that CD169+ cells were distributed evenly throughout the tissue, whereas CD169+CD163+ cells were located at the interface between FALCs and the surrounding adipose tissue (data not shown). Tim4+ CD163− cells were mainly located within FALCs, whereas CD163+ Tim4+ cells were only found at the interface between FALCs and the surrounding adipose tissue (data not shown), similarly to CD169+CD163+ cells (data not shown). Therefore, P1 (CD163+ Tim4+) and P2 (CD163+ Tim4-) macrophages are specifically located in the tissue surrounding FALCs, whereas P3 (CD163− Tim4−) macrophages are widely distributed throughout the adipose tissue, and P4 (CD163− Tim4+) macrophages appear to be located within FALCs.
[0163] To determine the dynamics of these macrophage subsets during colonization of omentum by ovarian cancer cells and tumor progression, we performed a kinetic analysis by flow cytometry after transplantation of ID8 cells. Among the CD11b+ myeloid fraction, CCR2+ monocytes represent the most abundant population in naïve omentum, which remained unchanged for up to 4 weeks after ID8 cell injection (
Conclusion
[0164] In summary, these studies showed that ID8 cells colonize the omentum in the vicinity of FALCs and juxtaposed to resident CD163+ Tim4+ macrophages. As tumors progressed, there was an increase in the proportion of CD169+ Lyve1+ macrophages, but the fraction of resident CD163+ Tim4+ cells remained stable.
Example 4—Embryonic Origin of Tissue-Resident CD163+ Tim4+ Macrophages in Omentum
[0165] To evaluate the ontogeny and homeostasis of the different macrophage subsets in omentum during ID8 tumor development, we prepared protected radiation chimeras using CD45.1 congenic C57BL/6 recipient mice. The abdomen of mice was protected with lead shielding to avoid radiation-induced replacement of tissue-resident macrophages. Following irradiation, we adoptively transferred bone marrow cells from CD45.1×CD45.2 F1 mice (CD45.1/.2), which allowed the distinction between host (CD45.1+) and donor cells (CD45.1+/CD45.2+) by flow cytometry (data not shown). Five weeks after the bone marrow transplantation, chimerism was confirmed in blood by flow cytometry (data not shown) before mice were injected with ID8 cells. After an additional 8 weeks of tumor growth omentum was harvested for analysis. To track bone marrow-derived cells, CD45.1 and CD45.2 expression by omental macrophages was analyzed and normalized to blood monocytes. Chimerism within P3 (CD163− Tim4−) and P4 (CD163− Tim4+) populations was on average close to 100%, in both naïve and tumor-bearing mice (
[0166] To verify the contribution of circulating monocytes to omental TAM, we next performed a fluorescent fate-mapping experiment using mice expressing Cx3crl-CreERT2 (Cx3crlCreER) and a Rosa26-lox-STOP-lox(LSL)-tdRFP reporter allele (Cx3crl-R26tdRFP) (Goossens et al., 2019; Yona et al., 2013). In adult mice, Cx3cr1 is expressed by monocytes in blood and omentum, but expression is decreased as monocytes mature into macrophages (data not shown). To label monocytes during tumor development, Cx3crl-R26tdRFP mice were injected with ID8 cells and after 6 weeks mice were given a single dose of tamoxifen by oral gavage to activate RFP expression in Cx3cr1+ cells. 10 days later, RFP in omental macrophages was assessed by flow cytometry. As expected, P3 and P4 populations were most strongly labeled with RFP under these conditions, whereas P2 macrophages were labeled to a lesser extent (
[0167] The experiments described above showed that CD163+ Tim4+ omental macrophages were not derived from bone marrow-dependent monocyte precursors, suggesting that these tissue-resident macrophages may be of embryonic origins. To evaluate the potential embryonic origins of these cells, we performed a fate mapping experiment with Cx3crlCreER mice in utero, since Cx3cr1 is expressed in both embryonic macrophage precursors and fetal monocytes (Yona et al., 2013). Cx3crl-R26YFP embryos were pulse-labeled with tamoxifen at E16.5 and YFP expression in macrophages from omentum was subsequently analyzed by flow cytometry at 8 weeks of age. After normalization of YFP+ cells to microglia to assess labeling efficiency (data not shown), we observed that approximately 20% of P1 macrophages (CD163+ Tim4+) in adult omentum were YFP+(
Conclusion
[0168] Collectively, these experiments suggest that CD163+ Tim4+ macrophages in omentum are of embryonic origin and uniquely independent of bone marrow-derived monocytes, both in steady-state and during tumor growth and are thus likely to maintain themselves locally by self-renewal.
Example 5—CD163+ Tim4+ Tissue-Resident Macrophages Express a Unique Transcriptional Profile
[0169] To evaluate the impact of tumor growth on the phenotype of different macrophage subsets in omentum, we performed transcriptional profiling of cells sorted by flow cytometry at steady-state and at different time points after seeding with tumor cells. We isolated P1, P2 and P3 macrophages by FACS from naïve omentum and at 5 or 10 weeks after injection of ID8 cells (data not shown). Sequencing libraries were prepared directly from snap-frozen cell pellets and sequenced to an average read depth of 42.7 million reads per sample. Expression values were normalized, filtered and analyzed for variations in gene expression using cqn and DeSeq2. A heatmap showing the 5000 most variable genes in the dataset is shown in figure S3. In order to analyze the relationship between the different macrophage populations, we performed a principal component analysis (PCA) combined with network analysis to show the n nearest neighbors. This analysis showed that the transcriptional profile of CD163+ Tim4+ resident macrophages (P1) did not undergo major changes during tumor growth, as all samples from this population clustered closely together (data not shown). Interestingly, the long-lived monocyte-derived CD163+ Tim4− macrophages (P2) were closely related to CD163+ Tim4+ resident macrophages (P1) at steady-state, but in tumor-bearing mice they became more closely aligned to P3 (data not shown), which likely reflects the increased replacement of P2 macrophages by monocyte-derived cells during tumor growth (data not shown). To further analyze the transcriptional changes between these three major subsets in established tumors, we extracted differentially expressed genes by pairwise and grouped comparisons of P1, P2 and P3 at 10 weeks of tumor growth (data not shown). We then performed a Self-Organizing-Map (SOM) clustering analysis to identify clusters of genes enriched in either a single population or a group of populations (data not shown). In the SOM clustering analysis, genes with a similar expression profile are first organized into SOMs. Within these maps, a pie chart depicts the relative enrichment of genes in the 3 populations analyzed (P1, P2 and P3) and the size of the pie slices reflects the gene enrichment. SOMs that are similar are then grouped together generating clusters of SOMs with a comparable gene enrichment profile. This analysis generated 16 different SOMs that were subsequently grouped in 7 distinct clusters. Cluster 2 was enriched in P1, cluster 6 in P2 and cluster 5 in P3. Cluster 3 contained genes enriched in both P1 and P2, and cluster 7 was enriched in both P2 and P3. We then used ClusterProfiler enrichment analysis and the Gene Ontology database (GO) for the different SOM clusters to identify biological processes (GO-BP) enriched in the different populations. This analysis revealed multiple enriched processes in clusters 2, 3, 5 and 6, the 15 most significant terms are shown in
Conclusion
[0170] This analysis suggests a functional diversification of omental macrophage subsets in the context of tumor growth.
Example 6—Specific Depletion of CD163+ Tim4+ Macrophages Prevents Metastatic Spread of Ovarian Cancer
[0171] Next, we sought to analyze the specific contributions of omental macrophage subsets to disease progression in our model. Initial experiments showed that tumor development and accumulation of malignant ascites were not affected in Ccr2−/− mice (data not shown). These mice have impaired recruitment of monocyte-derived cells, suggesting a redundant function of monocyte-derived macrophages in disease progression. To assess the specific contribution of CD163+ Tim4+ tissue-resident macrophages (P1), we generated transgenic mice that exclusively express DTR in CD163+ macrophages (Cd163-Csf1rDTR); Cd163-iCre knock-in mice (Cd163iCre) were crossed with transgenic mice expressing a LSL-DTR cassette under control of the Csflr-promoter (Tg(Csflr-LSL-DTR)). Flow cytometry analysis of omentum from Cd163-CsflrDTR mice 24 h after a single injection of diptheria toxin (DT; 4 ng/kg) confirmed the specific ablation of CD163+P1 and P2 macrophages (data not shown). However, 6 days after DT treatment, monocyte-derived P2 macrophages were completely restored while resident P1 macrophages remained absent (data not shown). Thus, this approach allows the specific ablation CD163+P1 macrophages and the opportunity to assess their unique contribution towards tumor progression. We treated cohorts of Cd163-Csf1rDTR and Cre-negative littermate controls (Csf1rLSL-DTR) with DT and 6 days later injected ID8 cells i.p. After 10 weeks, omentum was collected for analysis by flow cytometry and tumor progression assessed. At this time point, P1 macrophages remained depleted in omenta from Cd163-Csf1rDTR mice while all other populations were unchanged (
Conclusion
[0172] These data suggest tissue-resident CD163+ Tim4+ macrophages in the omentum contribute significantly to the metastatic spread of ovarian cancer cells and the development of invasive disease.
Example 7—the Role of CD163+ Macrophages in Tumor Progression
[0173] To further substantiate the role of CD163+ macrophages in tumor progression, we used CD163-targeted cytotoxic lipid nanoparticles (LNPs) to therapeutically deplete CD163+ macrophages in tumor-bearing mice (Etzerodt et al. 2019. The Journal of experimental medicine 216(10), 2394-2411.). These LNPs contain 5% polyethylene glycol (PEG; 2000 mw) in the lipid bilayer that minimizes non-specific phagocytic uptake and are loaded with the cytotoxic drug doxorubicin (dxr) to kill target cells. LNPs are targeted specifically to CD163-expressing cells by conjugation of an anti-CD163 monoclonal antibody to the PEG (Etzerodt et al., 2012). To achieve therapeutic depletion of CD163+ macrophages, mice were injected with ID8 cells and 5 weeks later treated intravenously twice a week for 5 weeks with either vehicle, empty CD163-targeted LNPs (αCD163-ctrl) or dxr-loaded LNPs (αCD163-dxr). In vivo bioluminescent imaging showed that continuous depletion of CD163+ cells after αCD163-dxr treatment, resulted in a significant reduction of overall tumor burden in the abdomen (
Conclusion
[0174] The data suggest that the absence of monocyte-derived CD163+ macrophages (P2) contribute to reduced tumor growth in the omentum (
Example 8—Ovarian Cancer Cells in Ascites Acquire Cancer Stem Cell (CSC) Characteristics
[0175] Ascitic tumor development and peritoneal metastases are characteristic of HGSOC and indicate a particularly poor prognosis. Our studies showed a long latency in ascitic tumor development and peritoneal spread of ID8 cells after seeding the omentum, and omentectomy prevented development of ascitic disease, suggesting the omentum represents a key pre-metastatic niche. In addition, the studies described above showed that tissue-resident macrophages in omentum contributed significantly to the accumulation of ascitic cells and the development of invasive disease. To further explore the invasive phenotype of ascitic ID8 cells and define the mechanisms behind this invasive transition, we performed transcriptomic analysis of cultured ID8 cells and tumor cells isolated from malignant ascites 11 weeks after transplantation (ID8-A11). With this analysis we found clusters of GO terms up-regulated in ID8-A11 cells that represent biological processes often associated with metastatic tumor cells, including; drug metabolism, epithelial cell migration, organization of cell junctions and organ development (data not shown). In contrast, clusters of GO terms that were down-regulated in ID8-A11 cells were mainly linked with biological processes associated with cell division such as cytokinesis, cell cycle, DNA repair and replication (data not shown). To further explore these pathways, we performed geneset enrichment analysis (GSEA); in accordance with a metastatic phenotype, GSEA showed a positive enrichment of genesets associated with epithelial to mesenchymal transition (EMT) and down-regulation of genes associated with cell cycle and cell division (data not shown). The inverse enrichment of EMT pathways versus cell division, coupled with the up-regulation of WNT, NOTCH and STAT3 signaling (data not shown), suggested ID8-A11 cells may have acquired a cancer stem cell (CSC)-like phenotype. Ascitic tumor cells with CSC characteristics have been associated with advanced human ovarian cancer, where they form multilayered spheroid structures (Bapat et al., 2005). To assess the CSC-like phenotype of ascitic ID8 cells, we compiled a list of genes that were reported as biomarkers of ovarian CSCs and analyzed their expression in ID8-A11 versus ID8 cells. This analysis confirmed an up-regulation of CSC markers in ID8-A11 cells (
Conclusion
[0176] These studies demonstrate that the transition to invasive disease in this model was associated with the acquisition of CSC characteristics by ascitic tumor cells.
Example 9—CD163+ Tim4+ Tissue-Resident Macrophages Promote the CSC-Like Phenotype Ovarian Cancer Cells
[0177] We next evaluated the impact of tissue-resident macrophages in omentum on the acquisition of CSC characteristics by ascitic ID8 cells. CD163+ macrophages in the omentum were depleted by 3 consecutive injections of CD163-targeted cytotoxic LNPs (αCD163-dxr) on days 1, 3, and 5, control mice were injected with either vehicle or empty LNPs (αCD163-ctrl). Monocyte-derived CD163+ macrophages (P2) were then allowed to recover before injection of ID8 cells on day 8 and the development of invasive disease was analysed at 10 weeks. Specific depletion of resident CD163+ Tim4+ macrophages (PO in omentum was confirmed by flow cytometry (
Conclusion
[0178] These experiments show that the acquisition of EMT and CSC characteristics by ID8 cells, and the development of invasive disease, is promoted by the resident CD163+ Tim4+ macrophages in the omentum.
Example 10—Treatment of Pancreatic Ductal Adenocarcinoma
Aim of Study
[0179] To further support the specific importance of CD163.sup.+ TAM in tumour progression, a CD163.sup.+ TAM depletion study in a GEMM model of pancreatic ductal adenocarcinoma (PDAC mice: P48-Cre; LSL-Kras.sup.G12D; LSL− Trp53.sup.R172H) was conducted.
Materials, Methods and Results
[0180] PDAC mice were treated with αCD163-dxr (1 mg/kg dxr) twice a week from 6 weeks of age; CD163.sup.+ TAM depletion resulted in increased survival with all mice still being alive at 20 weeks of age (
Conclusion
[0181] This example shows that pancreatic ductal adenocarcinoma can be treated and/or alleviated using a αCD163-dxr antibody. Thus, pancreatic cancer is indeed a target for the compositions, combinations and kits according to the invention.
Example 11—In Vitro Analysis of Duocarmycin Conjugated Anti-Mouse Tim4 Cytotoxicity
Aim of Study
[0182] To assess the in vitro cytotoxicity of duocarmycin conjugated anti-mouse Tim4.
Materials and Methods
Generation of Mouse Tim4 Expressing Cell Line:
[0183] cDNA coding for mouse Tim4 (NM 178759.4) was obtained from genscript and inserted in pcDNA5/FRT using HindIII and BamHI restriction sites. To generate a stable cell line expressing mouse Tim4, HEK FlpIn293 cells were co-transfected with pOG44 plasmid and Tim4_pcDNA/FRT plasmid and selected for Tim4 expression using 150 μg/ml Hygromycin. Stable expression of mouse Tim 4 was subsequent verified using western blotting against mouse Tim4 and image cytometry analysis.
Preparation of Anti-Mouse Tim4-Duocarmycin:
[0184] Anti-mouse Tim4 antibody (clone RMT4-54, BioXCell) or control IgG (rat IgG 2A, clone 2A3, BioXCell) was conjugated with OSu-PEG4-vc-PAB-Duocarmycin SA (Creative Biolabs) in a ratio of antibody to drug of 1:6 and a final antibody concentration of 1 mg/ml. pH was adjusted to 8.5 using Borate buffered saline pH 8.5 and reaction was left over night at 4° C. Non-conjugated duocarmycin was subsequently removed by dialysis overnight against 1×PBS at 4° C. resulting in an anti-mouse Tim4 antibody-drug conjugate with a DAR of 4.5 (0.86 mg/ml Tim4, 30.2 μM duocarmycin) and control IgG antibody-drug conjugate with a DAR of 4.2 (0.6 mg/ml antibody, 17 μM duocarmycin).
Results
[0185] FlpIn293 cells expressing mouse Tim4 was seeded in black clear bottom 96 well plates and incubated for 24 hrs. Following, 2 fold dilution (1 ug/ml to 1 ng/ml, each n=4) of duocarmycin conjugated anti-mouse Tim4 or control IgG was added to the cells. After 48 hrs, viability was measured using a calcein-AM based viability assay and relative viability was calculated using cells not receiving duocarmycin conjugated antibody (
Conclusion
[0186] Anti-mouse Tim4-duocarmycin could efficiently kill the Tim4 expressing cells in vitro.
Example 12—In Vivo Analysis of Duocarmycin Conjugated Anti-Mouse Tim4 Cytotoxicity and Specificity
Aim of Study
[0187] To assess the in vivo cytotoxicity of duocarmycin conjugated anti-mouse Tim4.
[0188] Materials and Methods
[0189] Naïve C57B1/6j were injected i.p with either vehicle (1×PBS, n=1) or 1 μg duocarmycin conjugated anti-mouse Tim4 (n=2) or 10 μg duocarmycin conjugated anti-mouse Tim4 (n=2). After 24 hrs mice were euthanized and peritoneal immune cells were harvested by peritoneal lavage using 1×PBS supplemented with 2 mM EDTA. Tim4 is mainly expressed by large peritoneal macrophages (LPM) and to assess cytotoxicity and specificity of duocarmycin conjugated anti-mouse Tim4 antibody, composition peritoneal immune cells were analyzed using flow cytometry. Peritoneal macrophages were gated as Live cells, CD45.2.sup.+, Ling.sup.neg (CD5, CD19, Ly6G, NK1.1), CD11.sup.+ and subsequently large peritoneal macrophages (LPM) were identified as F4/80.sup.+ MHCII.sup.+ whereas small peritoneal macrophages (SPM) were identified as F4/80.sup.− MHCII.sup.+ (data not shown). Subsequently frequency of Tim4 positive LPM and SPM were analyzed (
Results
[0190] The data in
Conclusion
[0191] This example confirms that an anti-mouse Tim4-duocarmycin antibody is specific and can efficiently deplete Tim4 expressing macrophages in vivo.
Discussion
[0192] The vast majority of cancer-related deaths are due to the development of metastatic disease. The metastatic spread of cancer can be described according to two basic models: The predominant linear model, dictates a step-wise progression of primary tumors before dissemination of fully metastatic malignant cells. Whereas the parallel model, accounts for the early dissemination of cancer cells and the formation of distant metastases that develop in parallel with the primary tumor. While there has been extensive research into the step-wise progression of primary tumors towards a metastatic phenotype, relatively little is known about the role of cells that form the pre-metastatic niche for disseminated cancer cells and their involvement in the metastatic spread of disease. Given that macrophages populate all adult tissues and the proven role of TAM in promoting invasion and metastasis in experimental models (Noy and Pollard, 2014), it's likely that resident macrophages form an important component of the tissue niche for the malignant progression of disseminated cancer cells.
[0193] Here we studied the origins and function of macrophages in a model of metastatic ovarian cancer using a novel intersectional transgenic approach and antibody-targeted cytotoxic liposomes to specifically deplete CD163+ macrophages. We describe how tissue-resident CD163+ macrophages in omentum play a specific role in the malignant progression of ovarian cancer and the development of invasive disease. We showed that ovarian cancer cells injected into the peritoneal cavity infiltrate the omentum in the vicinity of fat-associated lymphoid-clusters (FALC) and in close contact with resident CD163+ macrophages. Using flow cytometry and whole-mount imaging, we identified 4 distinct subsets of CD169+ Lyve-1+ mature macrophages in omentum based on their expression of CD163 and Tim4. CD163 has been used extensively as a marker of tissue macrophages in humans, where the frequency of CD163+ TAM shows a striking correlation with poor clinical outcome in a range of cancers (Komohara et al., 2014). Conversely, few studies have addressed the expression of Tim4 in human macrophages. Recent studies have shown Tim4 is expressed by a range of long-lived tissue-resident macrophages in the mouse, including Kupffer cells (Scott et al., 2016), large peritoneal macrophages (LPM) (Rosas et al., 2014), gut and cardiac macrophages (De Schepper et al., 2018; S. A. Dick et al., 2019), and is thus emerging as a marker of tissue-resident macrophages with potential for self-renewal. When analyzing the ontogeny of CD169+ Lyve-1+ macrophages in omentum, we found that Tim4 expression alone did not distinguish long-lived cells, since Tim4+ CD163− macrophages (P4), were monocyte-derived and rapidly replaced by bone marrow-derived cells. Whereas, CD163+ Tim4+ (P1) macrophages were of embryonic origin and uniquely independent of bone marrow-derived monocytes in the adult, in both steady-state and during tumor development. In contrast, CD163+ Tim4− macrophages (P2) were relatively long-lived at steady-state, compared to CD163-negative cells, but were quickly replaced by monocyte-derived cells after tumor initiation. Transcriptomic analysis revealed a close relationship between CD163+ P1 and P2 macrophages at steady-state, whereas, during tumor development monocyte-derived Tim4-negative P2 macrophages significantly diverged from Tim4+ P1 cells and became more similar to short-lived CD163-negative monocyte-derived cells (P3). In fact, the phenotype of CD163+ Tim4+ resident macrophages in omentum was remarkably stable during tumor progression, suggesting these cells maintain a level of tissue imprinting that is lost from more short-lived cells. Prophylactic depletion of CD163+ macrophages, allowing recovery of monocyte-derived CD163+ Tim4− cells, revealed an important and specific role for resident CD163+ Tim4+ omental macrophages in the development of invasive disease in this model. Furthermore, the therapeutic depletion of CD163+ TAM using cytotoxic liposomes had a major effect on tumor progression—illustrating the potential therapeutic implications for targeting TAM subsets in ovarian cancer.
[0194] Macrophages play important roles in organogenesis and tissue remodeling (Pollard, 2009) and have been shown to affect epithelial cell plasticity and stimulate tissue stem cells (Chakrabarti et al., 2018; Lee et al., 2018). The acquisition of stem-like characteristics by cancer cells (cancer stem cells; CSC) has been suggested to promote tumor progression and metastasis (Kreso and J. E. Dick, 2014). CSCs show increased anchorage-independent survival and high levels of resistance to chemotherapy or radiotherapy and thus have major implications for disease recurrence from disseminated tumor cells. EMT is also frequently associated with CSCs and accounts for the acquisition of migratory and invasive properties (Nieto et al., 2016). Several reports have shown that ascitic tumor cells from late-stage ovarian cancer patients show CSC-like characteristics (Bapat et al., 2005; Michela Lupia, 2017), which may explain the rapid progression of late-stage disease in these patients and the high frequency of disease recurrence. When analyzing the transcriptome of ascitic tumor cells in our model, we found a significant enrichment of pathways associated with CSCs and EMT. Interestingly, not only did the specific depletion of CD163+ Tim4+ resident macrophages in omentum decrease the formation of malignant ascites, it also reduced the frequency of CSCs among the few ascitic tumor cell that did accumulate. Analysis of tumor cells in the omentum showed that genes associated with regulation of EMT and CSCs, namely Stat3 (Abubaker et al., 2014), Wnt5a (Ford et al., 2014) and Mertk (Jung et al., 2016), were downregulated in tumor cells when CD163+ Tim4+ macrophages were absent, whereas Gata3, a negative regulator of EMT, was upregulated. In this regard, it is of particular interest that clustering analysis showed CD163+ Tim4+ macrophages were enriched for genes associated with positive regulation of the JAK-STAT pathway.
[0195] In summary, our data show that tissue-resident macrophages in omentum play a specific role in the malignant progression of disseminated tumor cells and the development of invasive disease in a mouse model of metastatic ovarian cancer. These studies add significantly to our understanding of TAM heterogeneity and the specific contribution of different macrophage subsets to disease progression. The axes of interaction between tissue-resident macrophages and cancer cells could represent important new therapeutic targets, not only in ovarian cancer but also other cancers where the development of CSCs can have a disastrous impact on disease prognosis.
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