SUMMARY 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 (https://github.com/multiplexIHC/cppipe) to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to thus improve biomarker discovery and assessment.

SUMMARY B cells foster squamous cell carcinogenesis (SCC) through deposition of immunoglobulin-containing immune complexes in premalignant tissue and Fcγreceptor-dependent activation of myeloid cells. Since human SCCs of the vulva and head and neck exhibited hallmarks of B cell infiltration, we examined B cell-deficient mice and found reduced ability to support SCC growth. Although ineffective as a single agent, treatment of mice bearing pre-existing SCCs with B cell-depleting αCD20 monoclonal antibodies improved response to platinum- and taxol-based chemotherapy. Improved chemo-responsiveness was dependent on altered chemokine expression by macrophages that fostered tumor infiltration of activated CD8+ T cells via CCR5-dependent mechanisms. These data reveal that B cells, and the downstream myeloid-based pathways they regulate, represent tractable targets for anti-cancer therapy in select tumors.

TGF-β and its signaling mediators, Smad2, -3, and -4, are involved with tumor suppression and promotion functions. Smad4 -/-mouse epidermis develops spontaneous skin squamous cell carcinomas (SCCs), and Smad3 -/-mice are resistant to carcinogen-induced skin cancer; however, the role of Smad2 in skin carcinogenesis has not been explored. In the present study, we found that Smad2 and Smad4, but not Smad3, were frequently lost in human SCCs. Mice with keratinocyte-specific Smad2 deletion exhibited accelerated formation and malignant progression of chemically induced skin tumors compared with WT mice. Consistent with the loss of Smad2 in poorly differentiated human SCCs, Smad2 -/-tumors were poorly differentiated and underwent epithelial-mesenchymal transition (EMT) prior to spontaneous Smad4 loss. Reduced E-cadherin and activation of its transcriptional repressor Snail were also found in Smad2 -/-mouse epidermis and occurred more frequently in Smad2-negative human SCCs than in Smad2-positive SCCs. Knocking down Snail abrogated Smad2 loss-associated EMT, suggesting that Snail upregulation is a major mediator of Smad2 loss-associated EMT. Furthermore, Smad2 loss led to a significant increase in Smad4 binding to the Snail promoter, and knocking down either Smad3 or Smad4 in keratinocytes abrogated Smad2 loss-associated Snail overexpression. Our data suggest that enhanced Smad3/Smad4-mediated Snail transcription contributed to Smad2 loss-associated EMT during skin carcinogenesis.

An mRNA differential display comparison of mouse JB6 promotionsensitive (P؉) and -resistant (P؊) cells identified a novel gene product that inhibits neoplastic transformation. The JB6 P؉ and P؊ cells are genetic variants that differ in their transformation response to tumor promoters; P؉ cells form anchorage-independent colonies that are tumorigenic, and P؊ cells do not. A differentially displayed fragment, A7-1, was preferentially expressed in P؊ cells at levels >10-fold those in P؉ cells, making its mRNA a candidate inhibitor of neoplastic transformation. An A7-1 cDNA was isolated that was identical to murine Pdcd4 gene cDNAs, also known as MA-3 or TIS, and analogous to human H731 and 197͞15a. Until now, the function of the Pdcd4 protein has been unknown. Paralleling the mRNA levels, Pdcd4 protein levels were greater in P؊ than in P؉ cells. Pdcd4 mRNA was also expressed at greater levels in the less progressed keratinocytes of another mouse skin neoplastic progression series. To test the hypothesis that Pdcd4 inhibits tumor promoter-induced transformation, stable cell lines expressing antisense Pdcd4 were generated from parental P؊ cells. The reduction of Pdcd4 proteins in antisense lines was accompanied by acquisition of a transformation-sensitive (P؉) phenotype. The antisense-transfected cells were reverted to their initial P؊ phenotype by overexpression of a Pdcd4 sense fragment. These observations demonstrate that the Pdcd4 protein inhibits neoplastic transformation.