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Immune checkpoint inhibitor (ICI) therapy has had limited success (<30%) in treating metastatic recurrent Head and Neck Oropharyngeal Squamous Cell Carcinomas (OPSCCs). We postulate that spatial determinants in the tumor play a critical role in cancer therapy outcomes. Here, we describe the case of a male patient diagnosed with p16 + OPSCC and extensive lung metastatic disease who failed Nivolumab and Pembrolizumab/Lenvatinib therapies. Using advanced integrative spatial proteogenomic analysis on the patient's recurrent OPSCC tumors we demonstrate that: (i) unbiased tissue clustering based on spatial transcriptomics (ST) successfully detected tumor cells and enabled the investigation of phenotypic traits such as proliferation or drug-resistance genes in the tumor's leading-edge and core; (ii) spatial proteomic imagining used in conjunction with ST (SpiCi, Spatial Proteomics inferred Cell identification) can resolve the profiling of tumor infiltrating immune cells, (iii) ST data allows for the discovery and ranking of clinically relevant alternative medicines based on their interaction with their matching ligand-receptor. Importantly, when the spatial profiles of ICI pre- and post-failure OPSCC tumors were compared, they exhibited highly similar PD-1/PD-L1 low and VEGFA high expression, suggesting that these new tumors were not the product of ICI resistance but rather of Lenvatinib dose reduction due to complications. Our work establishes a path for incorporating spatial-omics in clinical settings to facilitate treatment personalization., (© 2023. Nature Publishing Group UK.)

Available transcriptomes of the mammalian kidney provide limited information on the spatial interplay between different functional nephron structures due to the required dissociation of tissue with traditional transcriptome-based methodologies. A deeper understanding of the complexity of functional nephron structures requires a non-dissociative transcriptomics approach, such as spatial transcriptomics sequencing (ST-seq). We hypothesize that the application of ST-seq in normal mammalian kidneys will give transcriptomic insights within and across species of physiology at the functional structure level and cellular communication at the cell level. Here, we applied ST-seq in six mice and four human kidneys that were histologically absent of any overt pathology. We defined the location of specific nephron structures in the captured ST-seq datasets using three lines of evidence: pathologist's annotation, marker gene expression, and integration with public single-cell and/or single-nucleus RNA-sequencing datasets. We compared the mouse and human cortical kidney regions. In the human ST-seq datasets, we further investigated the cellular communication within glomeruli and regions of proximal tubules-peritubular capillaries by screening for co-expression of ligand-receptor gene pairs. Gene expression signatures of distinct nephron structures and microvascular regions were spatially resolved within the mouse and human ST-seq datasets. We identified 7,370 differentially expressed genes ( p adj < 0.05) distinguishing species, suggesting changes in energy production and metabolism in mouse cortical regions relative to human kidneys. Hundreds of potential ligand-receptor interactions were identified within glomeruli and regions of proximal tubules-peritubular capillaries, including known and novel interactions relevant to kidney physiology. Our application of ST-seq to normal human and murine kidneys confirms current knowledge and localization of transcripts within the kidney. Furthermore, the generated ST-seq datasets provide a valuable resource for the kidney community that can be used to inform future research into this complex organ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Raghubar, Pham, Tan, Grice, Crawford, Lam, Andersen, Yoon, Teoh, Matigian, Stewart, Francis, Ng, Healy, Combes, Kassianos, Nguyen and Mallett.)

Squamous cell carcinoma (SCC) is a common type of skin cancer that typically arises from premalignant precursor lesions named actinic keratoses (AK). Chronic inflammation is a well-known promoter of skin cancer progression. AK and SCC have been associated with an overabundance of the bacterium Staphylococcus aureus ( S. aureus ). Certain secreted products from S. aureus are known to promote cutaneous pro-inflammatory responses; however, not all S. aureus strains produce these. As inflammation plays a key role in SCC development, we investigated the pro-inflammatory potential and toxin secretion profiles of skin-cancer associated S. aureus . Sterile culture supernatants ("secretomes") of S. aureus clinical strains isolated from AK and SCC were applied to human keratinocytes in vitro. Some S. aureus secretomes induced keratinocytes to overexpress inflammatory mediators that have been linked to skin carcinogenesis, including IL-6, IL-8, and TNFα. A large phenotypic variation between the tested clinical strains was observed. Strains that are highly pro-inflammatory in vitro also caused more pronounced skin inflammation in mice. Proteomic characterization of S. aureus secretomes using mass spectrometry established that specific S. aureus enzymes and cytolytic toxins, including hemolysins, phenol-soluble modulins, and serine proteases, as well as currently uncharacterized proteins, correlate with the pro-inflammatory S. aureus phenotype. This study is the first to describe the toxin secretion profiles of AK and SCC-associated S. aureus , and their potential to induce a pro-inflammatory environment in the skin. Further studies are needed to establish whether these S. aureus products promote SCC development by mediating chronic inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Krueger, Zaugg, Chisholm, Linedale, Lachner, Teoh, Tuong, Lukowski, Morrison, Soyer, Hugenholtz, Hill and Frazer.)

Langerhans cells (LC) are skin-resident antigen-presenting cells that regulate immune responses to epithelial microorganisms. Human papillomavirus (HPV) infection can promote malignant epithelial transformation. As LCs are considered important for controlling HPV infection, we compared the transcriptome of murine LCs from skin transformed by K14E7 oncoprotein and from healthy skin. We identified transcriptome heterogeneity at the single cell level amongst LCs in normal skin, associated with ontogeny, cell cycle, and maturation. We identified a balanced co-existence of immune-stimulatory and immune-inhibitory LC cell states in normal skin that was significantly disturbed in HPV16 E7-transformed skin. Hyperplastic skin was depleted of immune-stimulatory LCs and enriched for LCs with an immune-inhibitory gene signature, and LC-keratinocyte crosstalk was dysregulated. We identified reduced expression of interleukin (IL)-34, a critical molecule for LC homeostasis. Enrichment of an immune-inhibitory LC gene signature and reduced levels of epithelial IL-34 were also found in human HPV-associated cervical epithelial cancers., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Prophylactic human papillomavirus (HPV) vaccines are commercially available for prevention of infection with cancerogenic HPV genotypes but are not able to combat pre-existing HPV-associated disease. In this study, we designed a nanomaterial-based therapeutic HPV vaccine, comprising manganese (Mn 4+ )-doped silica nanoparticles (Mn 4+ -SNPs) and the viral neoantigen peptide GF001 derived from the HPV16 E7 oncoprotein. We show in mice that Mn 4+ -SNPs act as self-adjuvants by activating the inflammatory signaling pathway via generation of reactive oxygen species, resulting in immune cell recruitment to the immunization site and dendritic cell maturation. Mn 4+ -SNPs further serve as Ag carriers by facilitating endo/lysosomal escape via depletion of protons in acidic endocytic compartments and subsequent Ag delivery to the cytosol for cross-presentation. The Mn 4+ -SNPs+GF001 nanovaccine induced strong E7-specific CD8 + T cell responses, leading to remission of established murine HPV16 E7-expressing solid TC-1 tumors and E7-expressing transgenic skin grafts. This vaccine construct offers a simple and general strategy for therapeutic HPV and potentially other cancer vaccines., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Human papillomaviruses infect keratinocytes and can lead to hyperproliferative dysplasia and malignant transformation if not cleared by the immune system. Human papillomavirus has evolved an array of mechanisms to evade and manipulate the immune system to improve replication efficiency and promote persistent infection. We here demonstrate that hyperproliferative skin expressing the high-risk human papillomavirus 16 E7 oncogene as a transgene drives immunomodulation of dendritic cells (DCs), resulting in reduced capacity to take up antigen and prime effector CD4 + T cell responses. The phenotype of DCs in the E7-expressing hyperproliferative skin was not reversible by activation through intradermal immunization. Naïve CD4 + T cells primed by E7-driven hyperproliferative skin acquired FoxP3 expression and an anergic phenotype. DC and T help modulation was dependent on E7-retinoblastoma protein interaction-driven epithelial hyperproliferation, rather than on expression of E7. Inhibition of binding of E7 to retinoblastoma protein, and of consequent epithelial hyperplasia, was associated with normal skin DC phenotype, and T helper type 1 effector responses to immunization were restored. We conclude that human papillomavirus-induced epithelial hyperplasia modulates epithelial DCs and inhibits T helper type 1 immunity while polarizing T-cell differentiation to a regulatory or anergic phenotype., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

"High-risk" human papillomaviruses (HPV) infect keratinocytes of squamous epithelia. The HPV16E7 protein induces epithelial hyperplasia by binding Rb family proteins and disrupting cell cycle termination. Murine skin expressing HPV16E7 as a transgene from a keratin 14 promoter (K14.E7) demonstrates epithelial hyperplasia, dysfunctional antigen presenting cells, ineffective antigen presentation by keratinocytes, and production of immunoregulatory cytokines. Furthermore, grafted K14.E7 skin is not rejected from immunocompetent non-transgenic recipient animals. To establish the contributions of E7, of E7-Rb interaction and of epithelial hyperplasia to altered local skin immunity, K14.E7 skin was compared with skin from K14.E7 mice heterozygous for a mutant Rb unable to bind E7 (K14.E7xRb ΔL/ΔL mice), that have normoplastic epithelium. Previously, we demonstrated that E7-speicfic T cells do not accumulate in K14.E7xRb ΔL/ΔL skin grafts. Here, we further show that K14.E7xRb ΔL/ΔL skin, like K14.E7 skin, is not rejected by immunocompetent non-transgenic animals. There were fewer CD11b + antigen presenting cells in skin draining lymph nodes from animals recipient of K14.E7xRb ΔL/ΔL grafts, when compared with animals receiving K14.E7 grafts or K5mOVA grafts. Maturation of migratory DCs derived from K14.E7xRb ΔL/ΔL grafts found in the draining lymph nodes is significantly lower than that of K14.E7 grafts. Surprisingly, K14.E7xRb ΔL/ΔL keratinocytes, unlike K14.E7 keratinocytes, are susceptible to E7 directed CTL-mediated lysis in vitro . We conclude that E7-Rb interaction and its associated epithelial hyperplasia partially contribute to the suppressive local immune responses in area affected by HPV16E7 expression.

Currently available vaccines prevent HPV infection and development of HPV-associated malignancies, but do not cure existing HPV infections and dysplastic lesions. Persistence of infection(s) in immunocompetent patients may reflect induction of local immunosuppressive mechanisms by HPV, providing a target for therapeutic intervention. We have proposed that a mouse, expressing HPV16 E7 oncoprotein under a Keratin 14 promoter (K14E7 mice), and which develops epithelial hyperplasia, may assist with understanding local immune suppression mechanisms that support persistence of HPV oncogene-induced epithelial hyperplasia. K14E7 skin grafts recruit immune cells from immunocompetent hosts, but consistently fail to be rejected. Here, we review the literature on HPV-associated local immunoregulation, and compare the findings with published observations on the K14E7 transgenic murine model, including comparison of the transcriptome of human HPV-infected pre-malignancies with that of murine K14E7 transgenic skin. We argue from the similarity of i) the literature findings and ii) the transcriptome profiles that murine K14E7 transgenic skin recapitulates the cellular and secreted protein profiles of high-grade HPV-associated lesions in human subjects. We propose that the K14E7 mouse may be an appropriate model to further study the immunoregulatory effects of HPV E7 expression, and can facilitate development and testing of therapeutic vaccines., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Chemokines regulate tissue immunity by recruiting specific subsets of immune cells. Mice expressing the E7 protein of human papilloma virus 16 as a transgene from a keratin 14 promoter (K14.E7) show increased epidermal and dermal lymphocytic infiltrates, epidermal hyperplasia, and suppressed local immunity. Here, we show that CXCL9 and CXCL10 are overexpressed in non-hematopoietic cells in skin of K14.E7 mice when compared with non-transgenic animals, and recruit CXCR3 + lymphocytes to the hyperplastic skin. Overexpression of CXCL9 and CXCL10 is not observed in E7 transgenic mice with mutated Rb gene whose protein product cannot interact with E7 (K14.E7xRb ΔL/ΔL ) and in consequence lack hyperplastic epithelium. CXCR3 + T cells are preferentially recruited by CXCL9 and CXCL10 in supernatants of K14.E7 but not K14.E7xRb ΔL/ΔL skin cultures in vitro. CXCR3 signalling promotes infiltration of a subset of effector T lymphocytes that enables donor lymphocyte deficient, E7-expressing skin graft rejection. Taken together, this suggests that recruitment of CXCR3 + T cells can be an important factor in the rejection of precancerous skin epithelium providing they can overcome local immunosuppressive mechanisms driven by skin-resident lymphocytes., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)