Abstract In the past 25 years, a majority of cancer studies have focused on examining functional consequences of activating and/or inactivating mutations in critical genes implicated in cell cycle ...control. These studies have taught us a great deal about the functions of oncogenes and tumor suppressor genes and the signaling pathways regulating cell proliferation and/or cell death. However, such studies have largely ignored the fact that cancers are heterogeneous cellular entities whose growth is dependent upon reciprocal interactions between genetically altered "initiated" cells and the dynamic microenvironment in which they live. This review highlights the aspects of cancer development that, like organogenesis during embryonic development and tissue repair in adult mammals, are regulated by interactions between epithelial cells, activated stromal cells, and soluble and insoluble components of the extracellular matrix.
Here, we describe a multiplexed immunohistochemical platform with computational image processing workflows, including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one ...formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination and revealed that response to therapy correlated with degree of mono-myelocytic cell density and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification and provide digital image processing pipelines to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to improve biomarker discovery and assessment.
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•Multiplex IHC and computational image analysis phenotypes tumor-immune complexity•In situ leukocyte density correlates with subclassification and prognosis in HNSCC•Immune complexity stratifies response to vaccination therapy in PDAC•CD8+ T cell and PD-L1 status correlate with outcomes of vaccinated PDAC patients
Tsujikawa et al. develop a multiplex immunohistochemistry and image cytometry platform to reveal immune-based metrics for patient stratification and response monitoring. In HNSCC and PDAC, prognosis correlates with mono-myelocytic cell density. In PDAC, percentages of PD-1, Eomes, Ki67, and granzyme B in CD8+ T cells correlate with response to vaccine therapy.
Leukocyte composition of human breast cancer Ruffell, Brian; Au, Alfred; Rugo, Hope S ...
Proceedings of the National Academy of Sciences - PNAS,
02/2012, Volume:
109, Issue:
8
Journal Article
Peer reviewed
Open access
Retrospective clinical studies have used immune-based biomarkers, alone or in combination, to predict survival outcomes for women with breast cancer (BC); however, the limitations inherent to ...immunohistochemical analyses prevent comprehensive descriptions of leukocytic infiltrates, as well as evaluation of the functional state of leukocytes in BC stroma. To more fully evaluate this complexity, and to gain insight into immune responses after chemotherapy (CTX), we prospectively evaluated tumor and nonadjacent normal breast tissue from women with BC, who either had or had not received neoadjuvant CTX before surgery. Tissues were evaluated by polychromatic flow cytometry in combination with confocal immunofluorescence and immunohistochemical analysis of tissue sections. These studies revealed that activated T lymphocytes predominate in tumor tissue, whereas myeloid lineage cells are more prominant in "normal" breast tissue. Notably, residual tumors from an unselected group of BC patients treated with neoadjuvant CTX contained increased percentages of infiltrating myeloid cells, accompanied by an increased CD8/CD4 T-cell ratio and higher numbers of granzyme B-expressing cells, compared with tumors removed from patients treated primarily by surgery alone. These data provide an initial evaluation of differences in the immune microenvironment of BC compared with nonadjacent normal tissue and reveal the degree to which CTX may alter the complexity and presence of selective subsets of immune cells in tumors previously treated in the neoadjuvant setting.
Intratumoral CD103+ dendritic cells (DCs) are necessary for anti-tumor immunity. Here we evaluated the expression of immune regulators by CD103+ DCs in a murine model of breast cancer and identified ...expression of TIM-3 as a target for therapy. Anti-TIM-3 antibody improved response to paclitaxel chemotherapy in models of triple-negative and luminal B disease, with no evidence of toxicity. Combined efficacy was CD8+ T cell dependent and associated with increased granzyme B expression; however, TIM-3 expression was predominantly localized to myeloid cells in both human and murine tumors. Gene expression analysis identified upregulation of Cxcl9 within intratumoral DCs during combination therapy, and therapeutic efficacy was ablated by CXCR3 blockade, Batf3 deficiency, or Irf8 deficiency.
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•TIM-3 is highly expressed by intratumoral CD103+ dendritic cells•TIM-3 antibody indirectly enhances a CD8+ T cell response during chemotherapy•TIM-3 antibody increases CXCL9 expression by dendritic cells•CXCL9 expression correlates with response to chemotherapy in breast cancer
de Mingo Pulido et al. show that intratumoral CD103+ dendritic cells (DCs) highly express TIM-3. Anti-TIM-3 antibody promotes CXCL9 expression by these DCs, which enhances the function of CD8+ T cells, thereby improving paclitaxel's therapeutic activity in breast cancer models.
B cells foster squamous cell carcinoma (SCC) development through deposition of immunoglobulin-containing immune complexes in premalignant tissue and Fcγ receptor-dependent activation of myeloid ...cells. Because human SCCs of the vulva and head and neck exhibited hallmarks of B cell infiltration, we examined B cell-deficient mice and found reduced support for SCC growth. Although ineffective as a single agent, treatment of mice bearing preexisting SCCs with B cell-depleting αCD20 monoclonal antibodies improved response to platinum- and Taxol-based chemotherapy. Improved chemoresponsiveness was dependent on altered chemokine expression by macrophages that promoted tumor infiltration of activated CD8+ lymphocytes via CCR5-dependent mechanisms. These data reveal that B cells, and the downstream myeloid-based pathways they regulate, represent tractable targets for anticancer therapy in select tumors.
•Human SCCs associated with high-risk HPV are infiltrated by Ig-producing B cells•B cell depletion or FcγR signaling inhibition impedes SCC neoplastic progression•Therapeutic B cell depletion enhances response of established SCCs to chemotherapy•B cell depletion reprograms macrophages to recruit CD8+ T cells to SCCs via CCR5
Affara et al. show that human squamous cell carcinomas (SCCs) associated with high-risk HPV are infiltrated by Ig-producing B cells. Therapeutic B cell depletion in mice bearing preexisting SCCs reprograms macrophages to recruit CD8+ T cells to SCCs via CCR5 and improves chemotherapeutic efficacy.
Most patients (80%) with ovarian cancer (OvCa) present with metastatic disease. Attachment of OvCa cells to peritoneum and omentum represents the first rate-limiting step for metastatic spread. ...Therefore, identifying factors regulating cell attachment in the abdominal cavity is critical to the development of therapeutic agents. We show here that MMP-2 expression was upregulated in OvCa cells upon attachment to their microenvironment. Downregulation of MMP-2 mRNA or pharmacological inhibition of MMP-2 proteolytic function, in both human OvCa primary cells and cell lines, reduced attachment of OvCa cells to a 3D organotypic model of metastatic OvCa, full human omentum or peritoneum, and in vivo to mouse peritoneum and omentum. Absence of MMP-2 in the host did not alter OvCa adhesion, as determined utilizing mice harboring homozygous null mutations in either the Mmp2 or Mmp9 genes. Conversely, adhesion induced upregulation of MMP-2 mRNA in OvCa cells. MMP-2 inhibition in OvCa cells through pharmacological or antibody treatment prior to i.p. dissemination in nude mice significantly decreased tumor growth and metastasis and extended survival. MMP-2 enhanced peritoneal adhesion of OvCa cells through cleavage of ECM proteins fibronectin (FN) and vitronectin (Vn) into small fragments and increased binding of OvCa cells to these FN and Vn fragments and their receptors, alpha5beta1 and alphaVbeta3 integrin. These findings indicate that MMP-2 expressed by metastatic OvCa cells functionally regulates their attachment to peritoneal surfaces.
Although abundant myeloid cell populations in the pancreatic ductal adenocarcinoma (PDAC) microenvironment have been postulated to suppress antitumor immunity, the composition of these populations, ...their spatial locations, and how they relate to patient outcomes are poorly understood.
To generate spatially resolved tumor and immune cell data at single-cell resolution, we developed two quantitative multiplex immunofluorescence assays to interrogate myeloid cells (CD15, CD14, ARG1, CD33, HLA-DR) and macrophages CD68, CD163, CD86, IFN regulatory factor 5, MRC1 (CD206) in the PDAC tumor microenvironment. Spatial point pattern analyses were conducted to assess the degree of colocalization between tumor cells and immune cells. Multivariable-adjusted Cox proportional hazards regression was used to assess associations with patient outcomes.
In a multi-institutional cohort of 305 primary PDAC resection specimens, myeloid cells were abundant, enriched within stromal regions, highly heterogeneous across tumors, and differed by somatic genotype. High densities of CD15
ARG1
immunosuppressive granulocytic cells and M2-polarized macrophages were associated with worse patient survival. Moreover, beyond cell density, closer proximity of M2-polarized macrophages to tumor cells was strongly associated with disease-free survival, revealing the clinical significance and biologic importance of immune cell localization within tumor areas.
A diverse set of myeloid cells are present within the PDAC tumor microenvironment and are distributed heterogeneously across patient tumors. Not only the densities but also the spatial locations of myeloid immune cells are associated with patient outcomes, highlighting the potential role of spatially resolved myeloid cell subtypes as quantitative biomarkers for PDAC prognosis and therapy.
Clinical and experimental data now clearly indicate that chronic inflammation significantly contributes to cancer development. Emerging out of these studies is an appreciation that persistent humoral ...immune responses exacerbate recruitment and activation of innate immune cells in neoplastic microenvironments where they regulate tissue remodeling, pro-angiogenic and pro-survival pathways that together potentiate cancer development. Population-based studies examining individuals with chronic inflammatory disorders have revealed that states of suppressed cellular immunity, in combination with enhanced humoral immunity and humoral immunity-associated cytokines, cooperate and effectively suppress anti-tumor immune responses while simultaneously enhancing angiogenesis and presumably overall cancer risk in afflicted tissue. In addition, studies in transgenic mouse models of de novo organ-specific cancer development have revealed that inflammation mediated by immunoglobulins and immune complexes might be functionally significant parameters of tumor promotion and progression. These recent advances support the hypothesis that enhanced states of local humoral and innate immune activation, in combination with suppressed cellular immunity and failed cytotoxic T cell anti-tumor immunity, alter cancer risk and therefore represent powerful targets for anti-cancer immunotherapeutics.
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