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• W4304196303 abstract "•Myeloid HCK expression is elevated in human pancreatic ductal adenocarcinoma•HCK inhibition, or its ablation in hosts, limits primary/metastatic PDAC growth•HCK ablation prevents an immune-suppressive and desmoplastic PDAC stroma•HCK inhibition enables and improves PDAC response to immunotherapy/chemotherapy Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a low 5-year survival rate and is associated with poor response to therapy. Elevated expression of the myeloid-specific hematopoietic cell kinase (HCK) is observed in PDAC and correlates with reduced patient survival. To determine whether aberrant HCK signaling in myeloid cells is involved in PDAC growth and metastasis, we established orthotopic and intrasplenic PDAC tumors in wild-type and HCK knockout mice. Genetic ablation of HCK impaired PDAC growth and metastasis by inducing an immune-stimulatory endotype in myeloid cells, which in turn reduced the desmoplastic microenvironment and enhanced cytotoxic effector cell infiltration. Consequently, genetic ablation or therapeutic inhibition of HCK minimized metastatic spread, enhanced the efficacy of chemotherapy, and overcame resistance to anti-PD1, anti-CTLA4, or stimulatory anti-CD40 immunotherapy. Our results provide strong rationale for HCK to be developed as a therapeutic target to improve the response of PDAC to chemo- and immunotherapy. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a low 5-year survival rate and is associated with poor response to therapy. Elevated expression of the myeloid-specific hematopoietic cell kinase (HCK) is observed in PDAC and correlates with reduced patient survival. To determine whether aberrant HCK signaling in myeloid cells is involved in PDAC growth and metastasis, we established orthotopic and intrasplenic PDAC tumors in wild-type and HCK knockout mice. Genetic ablation of HCK impaired PDAC growth and metastasis by inducing an immune-stimulatory endotype in myeloid cells, which in turn reduced the desmoplastic microenvironment and enhanced cytotoxic effector cell infiltration. Consequently, genetic ablation or therapeutic inhibition of HCK minimized metastatic spread, enhanced the efficacy of chemotherapy, and overcame resistance to anti-PD1, anti-CTLA4, or stimulatory anti-CD40 immunotherapy. Our results provide strong rationale for HCK to be developed as a therapeutic target to improve the response of PDAC to chemo- and immunotherapy. IntroductionPancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a 5-year survival rate of less than 10% (Raimondi et al., 2009Raimondi S. Maisonneuve P. Lowenfels A.B. Epidemiology of pancreatic cancer: an overview.Nat. Rev. Gastroenterol. Hepatol. 2009; 6: 699-708https://doi.org/10.1038/nrgastro.2009.177Crossref PubMed Scopus (464) Google Scholar). While chemotherapy confers transient tumor regression in 30% of patients, 9 out of 10 patients that undergo surgery still die of the disease due to local recurrence and/or metastasis (Conroy et al., 2011Conroy T. Desseigne F. Ychou M. Bouché O. Guimbaud R. Bécouarn Y. Adenis A. Raoul J.L. Gourgou-Bourgade S. de la Fouchardière C. et al.FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer.N. Engl. J. Med. 2011; 364: 1817-1825https://doi.org/10.1056/NEJMoa1011923Crossref PubMed Scopus (4987) Google Scholar). Likewise, immune checkpoint therapies including anti-(α)PD1 or αCTLA4 have failed to translate into meaningful improvements in a majority of PDAC patients (Diamond et al., 2021Diamond M.S. Lin J.H. Vonderheide R.H. Site-dependent immune escape due to impaired dendritic cell cross-priming.Cancer Immunol. Res. 2021; 9: 877-890https://doi.org/10.1158/2326-6066.Cir-20-0785Crossref PubMed Google Scholar; Galon and Bruni, 2019Galon J. Bruni D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies.Nat. Rev. Drug Discov. 2019; 18: 197-218https://doi.org/10.1038/s41573-018-0007-yCrossref PubMed Scopus (1167) Google Scholar).The poor response of PDAC to immune cell-related therapies can be accounted for by two major obstacles. The first tumor-intrinsic barrier relates to insufficient immune activation due to limited immunogenic mutations and presentation of cancer neo-epitopes, resulting in the current clinical recommendation for αPD1 to be limited as the second-line therapy for <1% of PDAC patients with DNA-mismatch repair deficient disease (Bailey et al., 2016Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. Miller D.K. Christ A.N. Bruxner T.J.C. Quinn M.C. et al.Genomic analyses identify molecular subtypes of pancreatic cancer.Nature. 2016; 531: 47-52https://doi.org/10.1038/nature16965Crossref PubMed Scopus (1889) Google Scholar; Rojas and Balachandran, 2021Rojas L.A. Balachandran V.P. Scaling the immune incline in PDAC.Nat. Rev. Gastroenterol. Hepatol. 2021; 18: 453-454https://doi.org/10.1038/s41575-021-00475-9Crossref PubMed Scopus (3) Google Scholar; Tempero et al., 2019Tempero M.A. Malafa M.P. Chiorean E.G. Czito B. Scaife C. Narang A.K. Fountzilas C. Wolpin B.M. Al-Hawary M. Asbun H. et al.Pancreatic adenocarcinoma, version 1.2019.J. Natl. Compr. Canc. Netw. 2019; 17: 202-210https://doi.org/10.6004/jnccn.2019.0014Crossref PubMed Scopus (220) Google Scholar). The second tumor-extrinsic barrier arises from an immunosuppressive and desmoplastic microenvironment characterized by an influx of cancer-associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs), which collectively promote the exclusion of cytotoxic T cells and natural killer (NK) effector cells from tumors (Ho et al., 2020Ho W.J. Jaffee E.M. Zheng L. The tumour microenvironment in pancreatic cancer — clinical challenges and opportunities.Nat. Rev. Clin. Oncol. 2020; 17: 527-540https://doi.org/10.1038/s41571-020-0363-5Crossref PubMed Scopus (282) Google Scholar). Thus, therapies that can simultaneously activate tumor immunity and relieve immune suppression represent promising adjuvant strategies to better control PDAC progression and metastasis.Elevated expression of the myeloid SRC family kinase hematopoietic cell kinase (HCK) is observed in most human solid malignancies including PDAC, where more than 95% of all HCK expression occurs in immune cells and correlates with poor patient survival (Bailey et al., 2016Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. Miller D.K. Christ A.N. Bruxner T.J.C. Quinn M.C. et al.Genomic analyses identify molecular subtypes of pancreatic cancer.Nature. 2016; 531: 47-52https://doi.org/10.1038/nature16965Crossref PubMed Scopus (1889) Google Scholar; Crnogorac-Jurcevic et al., 2005Crnogorac-Jurcevic T. Gangeswaran R. Bhakta V. Capurso G. Lattimore S. Akada M. Sunamura M. Prime W. Campbell F. Brentnall T.A. et al.Proteomic analysis of chronic pancreatitis and pancreatic adenocarcinoma.Gastroenterology. 2005; 129: 1454-1463https://doi.org/10.1053/j.gastro.2005.08.012Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar; Heidenblad et al., 2004Heidenblad M. Schoenmakers E.F.P.M. Jonson T. Gorunova L. Veltman J.A. van Kessel A.G. Höglund M. Genome-wide array-based comparative genomic hybridization reveals multiple amplification targets and novel homozygous deletions in pancreatic carcinoma cell lines.Cancer Res. 2004; 64: 3052-3059https://doi.org/10.1158/0008-5472.can-03-3159Crossref PubMed Scopus (0) Google Scholar; Isella et al., 2015Isella C. Terrasi A. Bellomo S.E. Petti C. Galatola G. Muratore A. Mellano A. Senetta R. Cassenti A. Sonetto C. et al.Stromal contribution to the colorectal cancer transcriptome.Nat. Genet. 2015; 47: 312-319https://doi.org/10.1038/ng.3224Crossref PubMed Scopus (417) Google Scholar; Poh et al., 2015Poh A.R. O'Donoghue R.J.J. Ernst M. Hematopoietic cell kinase (HCK) as a therapeutic target in immune and cancer cells.Oncotarget. 2015; 6: 15752-15771https://doi.org/10.18632/oncotarget.4199Crossref PubMed Scopus (72) Google Scholar; Zhu et al., 2021Zhu Z. Tang C. Xu T. Zhao Z. Molecular analysis of prognosis and immune pathways of pancreatic cancer based on TNF family members.J. Oncol. 2021; 2021: 2676996https://doi.org/10.1155/2021/2676996Crossref PubMed Scopus (1) Google Scholar). We have previously demonstrated a tumor-extrinsic role for myeloid HCK signaling in gastric and colon cancer by promoting an immunosuppressive tumor microenvironment (Poh et al., 2017Poh A.R. Love C.G. Masson F. Preaudet A. Tsui C. Whitehead L. Monard S. Khakham Y. Burstroem L. Lessene G. et al.Inhibition of hematopoietic cell kinase activity suppresses myeloid cell-mediated colon cancer progression.Cancer Cell. 2017; 31: 563-575.e5https://doi.org/10.1016/j.ccell.2017.03.006Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, Poh et al., 2020Poh A.R. Dwyer A.R. Eissmann M.F. Chand A.L. Baloyan D. Boon L. Murrey M.W. Whitehead L. O'Brien M. Lowell C.A. et al.Inhibition of the SRC kinase HCK impairs STAT3-dependent gastric tumor growth in mice.Cancer Immunol. Res. 2020; 8: 428-435https://doi.org/10.1158/2326-6066.CIR-19-0623Crossref PubMed Scopus (12) Google Scholar). Conversely, genetic ablation or pharmacologic inhibition of HCK reduced tumor growth (Poh et al., 2017Poh A.R. Love C.G. Masson F. Preaudet A. Tsui C. Whitehead L. Monard S. Khakham Y. Burstroem L. Lessene G. et al.Inhibition of hematopoietic cell kinase activity suppresses myeloid cell-mediated colon cancer progression.Cancer Cell. 2017; 31: 563-575.e5https://doi.org/10.1016/j.ccell.2017.03.006Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, Poh et al., 2020Poh A.R. Dwyer A.R. Eissmann M.F. Chand A.L. Baloyan D. Boon L. Murrey M.W. Whitehead L. O'Brien M. Lowell C.A. et al.Inhibition of the SRC kinase HCK impairs STAT3-dependent gastric tumor growth in mice.Cancer Immunol. Res. 2020; 8: 428-435https://doi.org/10.1158/2326-6066.CIR-19-0623Crossref PubMed Scopus (12) Google Scholar). Here, we establish the therapeutic benefit of targeting HCK in PDAC to reduce immune suppression, attenuate the desmoplastic response, and re-invigorate adaptive anti-tumor immunity.ResultsGenetic ablation of HCK in hosts reduces PDAC growth and metastasisTo explore a potential tumor-promoting role for HCK in pancreatic cancer, we interrogated the expression level of HCK in PDAC patients (Tang et al., 2017Tang Z. Li C. Kang B. Gao G. Li C. Zhang Z. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses.Nucleic Acids Res. 2017; 45: 98-102https://doi.org/10.1093/nar/gkx247Crossref PubMed Scopus (4623) Google Scholar) and observed elevated expression in tumor samples compared with matched normal tissues (Figure 1A ). We also analyzed single-cell RNA sequencing (scRNA-seq) datasets of human and mouse PDAC tumors (Elyada et al., 2019Elyada E. Bolisetty M. Laise P. Flynn W.F. Courtois E.T. Burkhart R.A. Teinor J.A. Belleau P. Biffi G. Lucito M.S. et al.Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts.Cancer Discov. 2019; 9: 1102-1123https://doi.org/10.1158/2159-8290.CD-19-0094Crossref PubMed Scopus (539) Google Scholar), and we confirmed that HCK was most prominently expressed in tumor-associated myeloid cells (Figures 1B and 1C).To determine whether aberrant myeloid HCK signaling in the host is involved in PDAC growth and metastasis, we orthotopically engrafted syngeneic KPC pancreatic tumor cells into the distal pancreas of wild-type (WT) and HCK knockout (HckKO) hosts, and we observed significantly smaller pancreatic tumors in HckKO hosts compared with their WT counterparts (Figures 1D and S1A). Moreover, HckKO hosts did not develop metastatic lesions, which we consistently observed in WT mice, including in the liver (n = 15/15), spleen (15/15), intestine (7/15), peritoneum (4/15), and kidneys (4/15) (Figure S1B). To obtain better insights into the contribution of HCK during PDAC metastasis, we injected KPC tumor cells into the spleen followed by splenectomy to protect hosts against premature death arising from overgrowth of the primary tumor. Again, we observed reduced incidence of liver metastasis in HckKO hosts (Figures 1E and S1A), and we confirmed in both orthotopic and intrasplenic PDAC models that Hck expression was restricted to the myeloid cell compartment of KPC tumors (Figure S1C). To formally prove that the enhanced anti-tumor response in HckKO hosts was an intrinsic consequence of hematopoietic cells lacking Hck expression, we generated reciprocal bone marrow chimeras and subjected these mice to the intrasplenic tumor model. We observed improved survival of WT←KO (Recipient←Donor) bone marrow chimeras compared with WT←WT hosts, and reduced survival of KO←WT hosts compared with KO←KO hosts (Figure S1D and Table S1). We also extended these observations to a therapeutic setting by treating WT mice with the HCK-specific small molecule inhibitor RK20449 (Saito et al., 2013Saito Y. Yuki H. Kuratani M. Hashizume Y. Takagi S. Honma T. Tanaka A. Shirouzu M. Mikuni J. Handa N. et al.A pyrrolo-pyrimidine derivative targets human primary AML stem cells in vivo.Sci. Transl. Med. 2013; 5: 181ra52https://doi.org/10.1126/scitranslmed.3004387Crossref PubMed Scopus (57) Google Scholar) after orthotopic or intrasplenic injection of KPC tumor cells. Compared with vehicle-treated mice, RK20449 treatment impaired the growth of primary tumors and liver metastases (Figures S1E and S1F).Genetic ablation of HCK enhances the immune-stimulatory endotype of myeloid cells and promotes an influx of activated cytotoxic effector cells into tumorsTo functionally link reduced Hck expression in myeloid cells to an improved anti-tumor immune response, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway analysis on bulk RNA sequenced KPC liver metastases recovered from WT and HckKO hosts. In tumors of HckKO hosts, we observed a significant enrichment of pathways associated with innate and adaptive immune responses, including myeloid cell activation and effector cell-mediated cytotoxicity (Figure 2A ).Figure 2Genetic ablation of HCK in myeloid cells enhances cytotoxic effector cell recruitment and activationShow full caption(A) Enriched KEGG and GO signaling pathways in KPC liver metastases of HckKO hosts compared with WT mice. n = 5 mice per group.(B) Flow cytometry quantification of myeloid cells in KPC liver metastases of WT and HckKO hosts. Each symbol represents an individual mouse. n = 20 mice per group.(C) Kaplan-Meier survival analysis of WT bone marrow chimeras reconstituted with cDC1-deficient (cDC1KO) or cDC1-proficient (WT) bone marrow. To deplete TAMs, half of each cohort were treated with αCSF1R prior to intrasplenic KPC tumor cell injection and continued until clinical endpoint. Following establishment of intrasplenic KPC tumors, mice were treated with the small molecule HCK inhibitor RK20449 or Captisol vehicle control. Shaded area indicates treatment period. n = 8 mice per group. A Mantel-Cox log rank test was used to evaluate statistical significance (see Table S3).(D) Kaplan-Meier survival analysis of tumor-bearing WT and HckKO hosts following NK cell, CD4+ T cell, or CD8+ T cell depletion. Shaded area indicates treatment period. n = 8 mice per group. A Mantel-Cox log rank test was used to evaluate statistical significance (see Table S5).(E) Flow cytometry quantification of CD8+ T cells and NK cells in KPC liver metastases of WT and HckKO hosts. Each symbol represents an individual mouse. n = 20 mice per group.(F) Quantification of granzyme B and perforin immunohistochemical staining in KPC liver metastases of WT and HckKO hosts. Each symbol represents an individual mouse. n = 10 mice per group. Data represent mean ± SEM; ∗∗∗p < 0.001, with statistical significance determined by an unpaired Student’s t test for comparison between two means. See also Figure S2 and Tables S3 and S5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Given the contribution of immunosuppressive myeloid cells to PDAC growth and metastasis (Poh and Ernst, 2021Poh A.R. Ernst M. Tumor-associated macrophages in pancreatic ductal adenocarcinoma: therapeutic opportunities and clinical challenges.Cancers. 2021; 13: 2860https://doi.org/10.3390/cancers13122860Crossref PubMed Scopus (7) Google Scholar), we next profiled myeloid cells in KPC liver metastases of WT and HckKO hosts and found that HCK deficiency did not affect the overall abundance of TAMs. Instead, we observed a reduction of both CD206+ alternatively activated macrophages and MDSCs, and an increase in CD103+ conventional type 1 dendritic cells (cDC1s) in tumors of HckKO hosts (Figure 2B). Importantly, DCs and TAMs from tumors of HckKO hosts displayed increased expression of immune-stimulatory factors (i.e., Il12α, Ifnγ, Tnf, Cxcl9, and Cxcl10) and a concomitant downregulation of genes associated with immune suppression (i.e., Il4, Il10, Il13, Tgfβ, Arg1) and matrix remodeling (i.e, Mmp7, Mmp9) (Figure S2A). Given that myeloid cells are the primary source of IL12 and CXCL9/CXCL10 (Arnold et al., 2019Arnold I.C. Zhang X. Artola-Boran M. Fallegger A. Sander P. Johansen P. Müller A. BATF3-dependent dendritic cells drive both effector and regulatory T-cell responses in bacterially infected tissues.PLoS Pathog. 2019; 15: e1007866https://doi.org/10.1371/journal.ppat.1007866Crossref PubMed Scopus (26) Google Scholar; Chow et al., 2019Chow M.T. Ozga A.J. Servis R.L. Frederick D.T. Lo J.A. Fisher D.E. Freeman G.J. Boland G.M. Luster A.D. Intratumoral activity of the CXCR3 chemokine System is required for the efficacy of anti-PD-1 therapy.Immunity. 2019; 50: 1498-1512.e5https://doi.org/10.1016/j.immuni.2019.04.010Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar; Garris et al., 2018Garris C.S. Arlauckas S.P. Kohler R.H. Trefny M.P. Garren S. Piot C. Engblom C. Pfirschke C. Siwicki M. Gungabeesoon J. et al.Successful anti-PD-1 cancer immunotherapy requires T cell-dendritic cell crosstalk involving the cytokines IFN-γ and IL-12.Immunity. 2018; 49: 1148-1161.e7https://doi.org/10.1016/j.immuni.2018.09.024Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar; House et al., 2020House I.G. Savas P. Lai J. Chen A.X.Y. Oliver A.J. Teo Z.L. Todd K.L. Henderson M.A. Giuffrida L. Petley E.V. et al.Macrophage-derived CXCL9 and CXCL10 are required for antitumor immune responses following immune checkpoint blockade.Clin. Cancer Res. 2020; 26: 487-504https://doi.org/10.1158/1078-0432.Ccr-19-1868Crossref PubMed Scopus (0) Google Scholar; Reschke and Gajewski, 2022Reschke R. Gajewski T.F. CXCL9 and CXCL10 bring the heat to tumors.Sci. Immunol. 2022; 7: eabq6509https://doi.org/10.1126/sciimmunol.abq6509Crossref PubMed Scopus (3) Google Scholar), we next determined the contribution of these molecules to HCK-dependent suppression of anti-tumor immunity using neutralizing antibodies against either IL12 or CXCR3 (cognate receptor for CXCL9/CXCL10). Blockade of IL12 or CXCR3 abrogated the survival benefit of HckKO hosts, while a difference in survival was not observed in WT hosts treated with these antibodies (Figure S2B and Table S2). Together, our findings suggest that genetic ablation of HCK promotes a shift of DCs and TAMs toward an activated endotype.We next assessed the contribution of cDC1s and TAMs to the enhanced anti-tumor response in HckKO hosts. Following reconstitution of lethally irradiated WT hosts with bone marrow from either cDC1-deficient (ItgaxCreIrf8fl/fl; Caton et al., 2007Caton M.L. Smith-Raska M.R. Reizis B. Notch-RBP-J signaling controls the homeostasis of CD8- dendritic cells in the spleen.J. Exp. Med. 2007; 204: 1653-1664https://doi.org/10.1084/jem.20062648Crossref PubMed Scopus (606) Google Scholar; Chopin et al., 2013Chopin M. Seillet C. Chevrier S. Wu L. Wang H. Morse III, H.C. Belz G.T. Nutt S.L. Langerhans cells are generated by two distinct PU.1-dependent transcriptional networks.J. Exp. Med. 2013; 210: 2967-2980https://doi.org/10.1084/jem.20130930Crossref PubMed Scopus (82) Google Scholar; Feng et al., 2011Feng J. Wang H. Shin D.M. Masiuk M. Qi C.F. Morse 3rd, H.C. IFN regulatory factor 8 restricts the size of the marginal zone and follicular B cell pools.J. Immunol. 2011; 186: 1458-1466https://doi.org/10.4049/jimmunol.1001950Crossref PubMed Scopus (59) Google Scholar; referred to as cDC1KO) or cDC1-proficient (WT) mice, we treated half of each cohort with a neutralizing antibody against CSF1R to also deplete TAMs prior to intrasplenic injection of KPC tumor cells. Following establishment of intrasplenic KPC tumors, mice were treated with the small molecule HCK inhibitor RK20449 or Captisol vehicle until clinical endpoint. While neither cDC1 nor TAM depletion affected the overall survival of vehicle-treated hosts compared with their immune cell-proficient controls, cDC1 depletion reduced the overall survival of RK20449-treated hosts, which was further reduced when TAMs were also simultaneously depleted (Figure 2C and Table S3).We then clarified the contribution of adaptive immunity to the enhanced anti-tumor response observed in HckKO hosts by exploiting the intrasplenic KPC model to establish liver metastasis in WT, HckKO, and lymphocyte-deficient Rag1KO and HckKO;Rag1KO compound mutant hosts. We observed enhanced liver tumor burden and reduced overall survival in HckKO;Rag1KO hosts compared with HckKO mice, as well as extended survival in HckKO hosts compared with all other cohorts (Figure S2C and Table S4). To delineate the role of NK and T cells in HckKO hosts, we individually depleted NK cells, CD4+ T cells, or CD8+ T cells, and we observed that CD8+ T cell depletion abrogated the tumor-suppressive effects conferred in the absence of HCK expression more effectively than either NK or CD4+ T cell depletion (Figure 2D and Table S5). These observations were consistent with an increased proportion of CD8+ T cells and NK cells in tumors of HckKO hosts compared with WT mice (Figures 2E and S2D). Furthermore, CD8+ T cells and NK cells isolated from tumors of HckKO hosts also showed elevated expression of genes encoding cytotoxic activities (i.e., Ifnγ, Tnf, GzmB, Prf1) (Figure S2E), and this in turn correlated with more abundant staining for granzyme B and perforin in tumors of HckKO hosts (Figure 2F). We surmised that these effects are primarily mediated by IL12 and CXCR3 signaling by myeloid cells, since administration of neutralizing antibodies against IL12 or CXCR3 significantly reduced CD8+ T cell recruitment and cytotoxicity in tumors of HckKO hosts (Figures S2F and S2G). Collectively, our findings suggest that CD8+ T cells are a major effector cell population that mediates the enhanced anti-tumor response in HckKO hosts.Genetic ablation of HCK in myeloid cells reduces the desmoplastic response of PDACCAFs produce and remodel most of the extracellular matrix (ECM) in PDAC (Geng et al., 2021Geng X. Chen H. Zhao L. Hu J. Yang W. Li G. Cheng C. Zhao Z. Zhang T. Li L. Sun B. Cancer-associated fibroblast (CAF) heterogeneity and targeting therapy of CAFs in pancreatic cancer.Front. Cell Dev. Biol. 2021; 9: 655152https://doi.org/10.3389/fcell.2021.655152Crossref PubMed Scopus (24) Google Scholar) and interact with myeloid cells to collectively amplify and sustain the immune-suppressive and fibrotic tumor microenvironment (Poh and Ernst, 2021Poh A.R. Ernst M. Tumor-associated macrophages in pancreatic ductal adenocarcinoma: therapeutic opportunities and clinical challenges.Cancers. 2021; 13: 2860https://doi.org/10.3390/cancers13122860Crossref PubMed Scopus (7) Google Scholar). Consistent with these observations, KPC liver metastases of HckKO hosts displayed significantly less ECM including collagen and fibronectin compared with their WT counterparts (Figure 3A ). We therefore examined whether CAFs associated with tumors of HckKO hosts differed quantitatively and qualitatively from those of tumors from WT hosts. Immunohistochemistry and flow cytometry revealed that tumors of HckKO hosts contained fewer inflammatory, myofibroblastic, and antigen-presenting CAFs (Figures 3B and 3C). Likewise, CAFs from HckKO hosts also displayed reduced expression of genes associated with immune suppression (i.e., Tgfβ, Il10), fibrosis (i.e., Il11), and ECM remodeling (i.e., Mmp3, Mmp7, Mmp9, Col1a1) than CAFs from tumors of WT hosts (Figure 3D).Figure 3Genetic ablation of HCK in myeloid cells reduces the desmoplastic tumor microenvironmentShow full caption(A) Representative immunohistochemical staining and quantification of ECM proteins in KPC liver metastases of WT and HckKO hosts. Collagen was visualized by Masson’s Trichrome (MT) staining. Scale bar: 100 μm. Each symbol represents an individual mouse. n ≥ 10 mice per group.(B) Representative immunohistochemical staining and quantification of pan-CAF markers in KPC liver metastases of WT and HckKO hosts. Scale bar: 100 μm. Each symbol represents an individual mouse. n ≥ 10 mice per group.(C) Flow cytometry quantification of CAFs in KPC liver metastases of WT and HckKO mice. iCAFs: inflammatory CAFs; mCAFs: myofibroblasts; apCAFs: antigen-presenting CAFs. Each symbol represents an individual mouse. n = 11 mice per group.(D) qPCR analysis on CD45−EpCAM–CD31−PDPN+PDGFRα+ CAFs isolated from KPC liver metastases of WT and HckKO mice. n = 5 mice per group. Data represent mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, with statistical significance determined by an unpaired Student’s t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Inhibition of HCK improves therapeutic response to chemotherapy and immunotherapyThe immune cell-excluded and fibrotic transcriptional profile of human PDAC correlates with poor clinical outcomes and resistance to immunotherapy (Bagaev et al., 2021Bagaev A. Kotlov N. Nomie K. Svekolkin V. Gafurov A. Isaeva O. Osokin N. Kozlov I. Frenkel F. Gancharova O. et al.Conserved pan-cancer microenvironment subtypes predict response to immunotherapy.Cancer Cell. 2021; 39: 845-865.e7https://doi.org/10.1016/j.ccell.2021.04.014Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Given that durable responses to αPD1 therapy are limited by the exhaustion of cytotoxic effector cells and an immunosuppressive and desmoplastic stroma, we first examined whether HCK deficiency sensitizes treatment-refractory PDAC tumors to αPD1. We treated tumor-bearing WT or HckKO hosts with αPD1, and we observed that genetic ablation of HCK in hosts enabled response to αPD1 immunotherapy and blocked the outgrowth of liver metastases (Figures 4A and S3A). This correlated with prolonged survival of all corresponding αPD1-treated HckKO mice well beyond that of all other treatment cohorts (Figure 4B and Table S6).Figure 4Genetic ablation of HCK in myeloid cells sensitizes PDAC tumors to immunotherapy and augments the efficacy of chemotherapyShow full caption(A) Representative whole mounts and corresponding liver weights of WT and HckKO hosts treated once every 3 days with αPD1 or a matched IgG isotype control. Treatment commenced on the 5th day after intrasplenic KPC tumor cell injection and continued for 2 weeks. Scale bar: 1 cm. Each symbol represents an individual mouse. n ≥ 15 mice per treatment group, n = 6 mice per treatment naive group.(B) Kaplan-Meier survival analysis of WT and HckKO hosts treated as described in Figure 4A until clinical endpoint. Shaded area indicates treatment period. n ≥ 10 mice per group. A Mantel-Cox log rank test was used to evaluate statistical significance (see Table S6).(C and E) Liver weights of WT and HckKO hosts treated once every 3 days with (C) αCTLA4, (E) αCD40, or a matched IgG isotype control. Treatment commenced on the 5th day after intrasplenic KPC tumor cell injection and continued for 2 weeks. Each symbol represents an individual mouse. n ≥ 11 mice per group.(D and F) Kaplan-Meier survival analysis of WT and HckKO hosts treated as described above with (D) αCTLA4, (F) αCD40, or a matched IgG isotype control until clinical endpoint. Shaded area indicates treatment period. n ≥ 10 mice per group. A Mantel-Cox log rank test was used to evaluate statistical significance (see Tables S7 and S8).(G) Liver weights of WT and HckKO hosts treated weekly with gemcitabine or PBS vehicle. Treatment commenced on the 5th day after intrasplenic KPC tumor cell injection and continued for 2 weeks. Each symbol represents an individual mouse. n ≥ 11 mice group.(H) Kaplan-Meier survival analysis of WT and HckKO mice treated as described in Figure 4G until clinical endpoint. Shaded area indicates treatment period. n ≥ 10 mice per group. A Mantel-Cox log rank test was used to evaluate statistical significance (see Table S9). Data represent mean ± SEM; ∗∗∗p < 0.001, with statistical significance determined by one-way ANOVA followed by Tukey’s multiple comparison test or Mantel-Cox log rank test for Kaplan-Meier analysis. See also Figures S3 and S4 and Tables S6–S9.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether HCK inhibition not only alleviated αPD1-mediated local immunosuppression but also stimulated antigenic priming, we next assessed the contribution of HCK ablation in the host to therapeutic CTLA4 inhibition or CD40 stimulation, respectively. Unlike the PD1 checkpoint, which suppresses T cell activatio" @default.
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