Although cell migration has been extensively investigated using in vitro model systems, the mechanisms underlying mammalian cell migration in native tissue environments remain underexplored. Moreover, efforts to directly manipulate and visualize molecular regulators in live mammalian tissues have been scarce. In this article, we first review the current insights into various single-cell migration phenomena, including stem cell types, observed in mammalian tissues under homeostatic and pathophysiological conditions. Thereafter, we discuss intravital subcellular microscopy (ISMic) as a tool to unravel membrane remodeling mechanisms underlying cell migration in live animal tissues. Lastly, we emphasize the need for innovative microscopy and complementary advanced approaches to achieve a deeper fundamental understanding of cell migration modalities and their impact on mammalian tissue in homeostasis and pathophysiology.

Directional persistence is essential for efficient immune cell migration in tissues, yet how cytoskeletal systems stabilize migration in complex three-dimensional environments remains unclear. Using intravital subcellular microscopy and quantitative analysis of membrane dynamics, we identify two spatially distinct architectures of non-muscle myosin II (NMII) that coordinate protrusion dynamics during neutrophil migration. In vivo and in collagen matrices, NMII assembles at the leading edge into lattice-like structures that are structurally and functionally distinct from rear contractile actomyosin bundles. Protrusion-resolved analyses reveal that directional persistence correlates strongly with protrusion lifetime and sustained NMII engagement, with rear NMII load showing the strongest association with protrusion persistence. Strikingly, directional migration is not determined by the abundance of favorable protrusions but by their temporal organization during migration. Pharmacological perturbations that redistribute NMII activity disrupt this temporal organization and alter migration trajectories. Together, these findings reveal that spatially distinct NMII architectures coordinate protrusion dynamics across time to stabilize directional migration in complex environments.

List and information of antibodies used in this study

Abstract Tumor progression is driven by cancer cells’ ability to establish a cellular network through tunneling nanotube-like connections (TNTs), which enable mitochondrial exchange both within the tumor cells and with the tumor microenvironment (TME). However, the effects of mitochondrial transfer between tumor and non-tumor cells, and its occurrence in vivo , remain poorly understood. In this study, we demonstrate bidirectional mitochondrial transfer: damaged mitochondria from Glioblastoma (GBM) cells trigger mitophagy in non-tumoral astrocytes (AS), while healthy mitochondria from AS enhance the metabolic activity of GBM cells. Furthermore, intravital subcellular microscopy (ISMic) in a live animal model, allowed the visualization of TNT connections with characteristics similar to those observed in vitro and supported TNT-mediated mitochondrial transfer in vivo . These findings provide critical insights into TME interactions and their contribution to cancer resilience, highlighting TNTs as a potential target for therapeutic intervention and paving the way for further research into their role in cancer.

Development of BCC cell line, effect of BAY60-6583 in human GLI and expression of Gs-coupled-GPCRs in single cell clusters

Understanding how cells regulate plasma membrane architecture inside intact living organs in a live animal has been limited by the inability to directly measure molecular dynamics in vivo. Here we introduce intravital single-molecule microscopy (iSiMM), an imaging approach that enables tracking of individual, endogenously expressed cytoskeletal components at the plasma membrane in live mice. Applying iSiMM to murine acinar secretory cells, we identify discrete basolateral membrane domains built on deeply folded membrane infolds that function as a pre-existing membrane reservoir. Single-molecule measurements reveal continuous, regulated molecular turnover within these domains. Physiological stimulation accelerates cytoskeletal exchange promoting rapid membrane unfolding and cell expansion. Together, these findings establish iSiMM as a general strategy for probing molecular kinetics underlying dynamic cellular behaviors in intact organs. One-sentence summary:Intravital single-molecule microscopy enables direct measurement of molecular kinetics underlying dynamic cellular behaviors in intact living organs.

Abstract Cancer cells exploit the intricate network of stimulatory and inhibitory pathways to prevent immune-mediated clearance and thrive in an immunocompetent biological system. While targeting immune checkpoints (ICs) to reactivate the immune response has proven to enhance tumor-immune infiltration and patient prognosis, efficacy is limited only to a subset of patients. Hence, identifying alternative approaches to enable anti-tumor immune response is crucial. Aberrant protein glycosylation is a hallmark of cancer; in particular, increased cell-surface sialylation has been shown to inhibit immune-mediated clearance of tumor cells. This is mediated by the sialic-acid-binding immunoglobulin-like lectins (Siglecs), a family of surface receptors that mediate inhibitory signals by binding to sialic acid. Several factors complicate targeting the Sialic acid-Siglec axis in cancer therapy, including (1) the systemic adverse effects of broad abrogation of sialylation or Siglecs binding, (2) the diverse binding affinities of Siglecs based on sialic acid linkage, scaffold glycan and carrying protein. To enable the full use of this regulatory pathway, we set out to determine how the sialoglycome is dysregulated during early tumorigenesis. To this end, we used longitudinal intravital microscopy to record premalignant growth within the same animal at cellular resolution over 24 weeks in a carcinogen-induced model of head and neck squamous cell carcinoma (HNSCC). This allowed us to stratify lesions into progressing, regressing and stable growth signatures. We employ spatially resolved glycomics, transcriptomics and multiplex staining to generate a longitudinal signature of premalignant lesions. We observe an increased abundance of glycans decorated with α2,3-linked sialic acid (α2,3Sia). The elevated levels of α2,3Sia reflected a significant upregulation of α2,3-Sialyltransferases expression, indicating control mechanisms at the transcriptional levels. In the epithelial compartment, α2,3Sia was particularly enriched in CD44+ de-differentiated tumor cells. Using Siglec-Fc Chimeric constructs, we observed a binding preference of the CD44+ tumor-cells to Siglec-E. α2,3Sia was also elevated in the lesion stroma, strikingly in ly6G+ CD11b+ neutrophils recruited to progressing lesions. Our findings suggest a potential engagement of the Siglec-E - α2,3Sia axis in premalignant HNSCC lesions. Ongoing analysis is dissecting the tumor-immune interactions implicated in this axis as a potential regulator of the immune spatio-temporal landscape during early tumorigenesis. This study offers a groundwork to delineate glycomic and transcriptomic signatures in premalignant lesions with the opportunity to identify potential preventative measures in harnessing the immune response in patients. Citation Format: Sarah M. Hammoudeh, Thomas D. Madsen, Roberto Weigert. Resolving the initiating changes in the immunomodulatory glycome during early tumorigenesis of head and neck squamous cell carcinoma in living animals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2252.

Basal cell carcinoma (BCC), the most common human cancer, is driven by hyperactivation of the Hedgehog pathway mediated by Smoothened (SMO) signaling and Glioma-Associated Oncogene Homolog (GLI) transcription. Gαs and protein kinase A (PKA) negatively regulate Hedgehog signaling, offering a potential alternative BCC development and treatment pathway. In this study, using histology alongside bulk and single-cell RNA sequencing, we found that mouse BCC-like tumors that originate from Gαs pathway inactivation are highly similar to those driven by canonical Hedgehog signaling induced by constitutive SMO activation. Both pathways led to the expansion of basal stem cells in the skin, with tumor cells clustering in two distinct populations with markers for touch dome and isthmus stem cell-like cells. Interestingly, mutations that reduce Gαs and PKA activity were present in human BCC. Tumors from Gαs pathway inactivation were independent of the canonical Hedgehog regulators SMO and GPR161, establishing them as SMO-independent oncogenic Hedgehog signaling models. Finally, activation of the Gαs-coupled adenosine 2B receptor with BAY60-6583 counteracted oncogenic SMO, reducing Hedgehog signaling and tumor growth. Together, these findings offer a potential therapeutic strategy for BCC. SIGNIFICANCE:Gαs/PKA pathway inactivation drives Hedgehog-dependent basal cell carcinoma and can be counteracted by activation of the Gαs-coupled adenosine 2B receptor to suppress tumor growth, providing a potential treatment for Hedgehog-driven tumors.

List and information of primers used in this study

mRNA localization to specific subcellular regions is common in mammalian cells but poorly understood in terms of its physiological roles. This study demonstrates the functional importance of Net1 mRNA, which we find prominently localized at the dermal-epidermal junction (DEJ) in stratified squamous epithelia. Net1 mRNA accumulates at DEJ protrusion-like structures that interact with the basement membrane and connect to a mechanosensitive network of microfibrils. Disrupting Net1 mRNA localization in mouse epithelium alters DEJ morphology and keratinocyte-matrix connections, affecting tissue homeostasis. mRNA localization dictates the cortical accumulation of the Net1 protein and its function as a RhoA GTPase exchange factor (GEF). Altered RhoA activity is in turn sufficient to alter the ultrastructure of the DEJ. This study provides a high-resolution in vivo view of mRNA targeting in a physiological context. It further demonstrates how the subcellular localization of a single mRNA can significantly influence mammalian epithelial tissue organization, thus revealing an unappreciated level of post-transcriptional regulation that controls tissue physiology.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic that induces apoptosis in cancer cells while sparing the non-malignant cells in preclinical models. However, its efficacy in clinical trials has been limited, suggesting unknown mechanisms modulating TRAIL activity in patients. We hypothesized that TRAIL treatment elicits transcriptional changes in triple negative breast cancer (TNBC) cells that alter the immune milieu. RNAseq analysis of MDA-MB-231 cells along with validation in additional cell lines demonstrated that TRAIL induced cytokines such as CXCLs 1, 2, 3, 8,11 and IL-6, which are known to modify neutrophil function. Mechanistically, TRAIL dependent induction of the cytokines was predominantly mediated by death receptor 5, caspase-8 and the non-canonical NFKB2 pathway. These cytokines produced by TRAIL-treated TNBC cells enhanced chemotaxis of normal human donor isolated neutrophils. Using TNBC xenograft models, TRAIL induced activation of NFkB2 pathway, cytokine production and increased neutrophil recruitment into the tumors. Moreover, preincubation of neutrophils in supernatants from TRAIL-treated TNBC cells significantly impaired neutrophil function as measured by reduced respiratory burst and cytotoxic effect against TNBC cells. Transcriptomic analysis of neutrophils incubated with either TRAIL alone or supernatant of TRAIL-treated TNBC cells revealed increased expression of inflammatory cytokines, immune modulatory genes, immune checkpoint genes, and genes implicated in delayed neutrophil apoptosis. Functional studies showed that these neutrophils suppress T cell proliferation and augment Treg suppressive phenotype. Collectively, our study demonstrates a novel role of TRAIL-induced NFKB2-dependent cytokine production that promotes neutrophil chemotaxis and neutrophil-mediated immune suppression.

Glycolipid-lectin-driven endocytosis controls the formation of clathrin-independent carriers and the internalization of various cargos such as beta 1 integrin. Whether this process is regulated in a dynamic manner remained unexplored. Here we demonstrate that, within minutes, the epidermal growth factor triggers the galectin-driven endocytosis of cell-surface glycoproteins, such as integrins, that are key regulators of cell adhesion and migration. The onset of this process-mediated by the Na+/H+ antiporter NHE1 as well as the neuraminidases Neu1 and Neu3-requires the pH-triggered enzymatic removal of sialic acids whose presence otherwise prevents galectin binding. De-sialylated glycoproteins are then retrogradely transported to the Golgi apparatus where their glycan make-up is reset to regulate EGF-dependent invasive-cell migration. Further evidence is provided for a role of neuraminidases and galectin-3 in acidification-dependent bone resorption. Glycosylation at the cell surface thereby emerges as a dynamic and reversible regulatory post-translational modification that controls a highly adaptable trafficking pathway.

The spontaneous regression of cancer lesions demonstrates the potential of immune surveillance; yet, these transient events have remained inaccessible to systematic study. Using longitudinal intravital microscopy in a carcinogen-induced model of head and neck cancer, we tracked premalignant lesions within the same animals for 24 weeks at single-cell resolution. This approach revealed three trajectories: progression, stability, or regression, and enabled dissection of the immune dynamics underlying each fate. Lesion outcome was dictated by the spatial organization of myeloid-derived antigen-presenting cells: regressing lesions were characterized by dense clusters of myeloid-derived cells associated with CXCL9+/CXCL10+ expression and T cell recruitment, whereas progressing lesions displayed sparse, non-clustered infiltration. Remarkably, transient myeloid clusters arose prior to any detectable lesion formation and consistently marked regions that would later develop into premalignant lesions. These findings identify spatiotemporal myeloid organization as an early determinant of tumor fate and provide a mechanistic framework for predicting and intercepting cancer at its inception.

Treatment resistance and relapse remain significant challenges in cancer therapy including chemotherapy, radiation, small-molecule inhibitors, and immunotherapy. While these treatments induce considerable apoptotic cell death, the complete range of apoptotic signals and their "off-target effects" on surviving cancer cells are not well understood. Our previous studies uncovered that apoptotic cancer cells with high levels of Peptidyl Arginine Deiminase 4 (Padi4) undergo nuclear expulsion, releasing DNA/protein complexes (Nuclear Expulsion Products; NEPs) into the extracellular space, which are critical for metastatic outgrowth. Here, we report that chemotherapy and radiotherapy trigger nuclear expulsion in Padi4-positive tumors, which in turn facilitates therapy resistance. Immune profiling revealed increased CD8+ T cell infiltration and activation in Padi4high tumors, which is characterized by elevated expression of PD1, GrnzB, IFNg, and Ki-67. Cisplatin treatment further amplified these responses, ultimately leading to CD8+ T cell exhaustion, exhibiting TIM3. Notably, CD8+ T cells were shown to be essential for nuclear expulsion-mediated therapy resistance, as demonstrated by experiments using CD8 neutralizing antibodies. Proteomic analysis and coculture assays identified extracellular citrullinated vimentin (citVIM) as a major NEP component, acting as a Damage-Associated Molecular Pattern (DAMP), which directly facilitated CD8+ T cell hyperactivation and contributed to rapid exhaustion. Through co-culture assays using TLR4-deficient CD8+ T cells and NEPs, we further demonstrated that TLR4 serves as a crucial binding partner of vimentin, linking NEP-derived vimentin to CD8+ T cell activation. In addition, citVIM was observed to enhance IFNγ production in CD8+ T cells, and IFNγ-primed 4T1 cells also demonstrated increased therapy resistance. These findings underscore CD8+ T cell-derived IFNγ as a critical downstream effector in therapy resistance and highlight the pivotal role of the citVIM-TLR4-IFNγ axis in nuclear expulsion-mediated chemoresistance. Collectively, our study reveals how apoptotic signals from cancer cells during therapy act as off-target effects, modulating host immunity, and demonstrate how cancer cells exploit these signals for survival. Furthermore, by elucidating the relationship between nuclear expulsion and T cells, this research provides a foundation for understanding how chemo- and radiotherapy could enhance the efficacy of immune checkpoint inhibitors (ICIs) Woo-Yong Park, Rachel Yang, Jae young So, Abdul Ahad, Yeap Ng, Balamurugan kuppusamy, Ronald holewinski, Roberto Weigert, Esta Sterneck, Thorkell Andresson, Li Yang. Nuclear expulsion-derived extracellular vimentin accelerates CD8+ T cell dysfunction and enhances treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3782.

Aneuploidy, a cancer hallmark, drives chromosomal instability, drug resistance, and clinically aggressive tumors. Cyclin-dependent kinase 2 (CDK2) antagonism with independent inhibitors or CDK2 knockdown triggered anaphase catastrophe. This disrupts supernumerary centrosome clustering, causing multipolar division and apoptosis. Time-lapse fluorescence microscopy of fluorescent ubiquitination-based cell cycle indicator (FUCCI) cell cycle probes transduced into aneuploid lung cancer cells revealed distinct fates of bipolar and polyploid cells after CDK2 inhibition. Apoptosis occurred in multipolar progeny but was repressed in persistent polyploid cancer cells. RNA-Seq analyses after CDK2 inhibition of 4N versus 2N lung cancer cells were enriched for CDK1 pathway and KIF family members. The Cancer Genome Atlas (TCGA) analysis of lung cancers indicated that CDK1 and KIF family member overexpression was associated with an unfavorable survival. Intravital microscopy of transplanted lung cancer cells in mice extended findings from the in vitro to in vivo settings. CDK2 inhibition of tumor-bearing mice produced polyploid cancer cells in vivo. These cancer cells were resistant to apoptosis and proliferated despite CDK2 inhibition. In contrast, polyploid populations were rarely detected in CDK2-inhibited human alveolar epithelial cells. These findings are translationally relevant. Combined targeting of CDK2 with CDK1 or kinesin family member antagonists should eliminate polyploid cancer cells, promote apoptosis, and augment antineoplastic effects.

The glycan makeup of membrane glycoproteins and glycosphingolipids at the cell surface is traditionally viewed as mature and static. Recent findings challenge this view, showing that selective glycan remodeling can redirect membrane glycoproteins back to the Golgi for another go. In this review we discuss the glycosylation processes in cells, with a focus on the terminal glycan chains on proteins and lipids that are capped by sialic acid sugars, and that engage the glycan-binding proteins of the galectin family. We highlight new studies demonstrating that growth factors trigger the removal of sialic acid by endogenous neuraminidases at the cell surface, leading to glycolipid-lectin driven endocytosis and retrograde traffic to the Golgi. This molecular circuit, termed the GlycoSwitch, introduces new perspectives on glycan-mediated regulation of cellular functions.

Aneuploidy, a hallmark of cancer, drives chromosomal instability and antineoplastic drug resistance. Common in hematologic and solid tumors, centrosome amplification leads to supernumerary centrosomes, causing chromosomal missegregation in dividing cancer cells. This imbalance produces progeny with abnormal chromosome counts, leading to cell death, resistance to antineoplastics, and clinically-aggressive tumors. This study examined the effects of CDK2 inhibition on aneuploid lung cancer cells and tumors. It revealed that CDK2 antagonism triggers anaphase catastrophe through centrosome clustering disruption and multipolar mitosis. Unexpectedly, this also leads to the formation of a persistent population of polyploid, apoptosis-resistant cells. Using independent CDK2 inhibitors and shRNAs, we observed that some cancer cells underwent multipolar division and subsequent cell death. Others exhibited cytokinesis failure, resulting in multinucleated, and polyploid cancer cells that continued to survive and proliferate despite continuous CDK2 inhibition. Through time-lapse imaging with transfected FUCCI cell cycle probes and RNA-seq analysis of polyploid versus diploid populations, we identified upregulated CDK1-related pathways and kinesin (KIF) family members in the polyploid cellular population. This likely contributes to the persistent polyploid population, despite CDK2 antagonism. These findings were independently validated in vivo using intravital microscopy and patient-derived xenograft (PDX) lung cancer models. Intriguingly, polyploid and multinucleated cancer cells were found to proliferate post-CDK2 inhibitor treatments. In marked contrast, a polyploid population was not found in CDK2 inhibited primary human alveolar epithelial cells, indicating that this is a pathway that is preferentially activated in aneuploid cancer cells. Analysis of gene expression in TCGA lung cancer datasets linked CDK1 and KIF family overexpression to an unfavorable patient survival, implicating this persistent polyploid population as contributing to poor survival outcomes in lung cancer. These findings are relevant to the clinical development of CDK2 inhibitors. They indicate that despite the pro-apoptotic effects of CDK2 antagonism in aneuploid lung cancer cells, a population of drug-resistant, polyploid cells persists. We propose that a combinatorial therapy targeting CDK2 alongside polo-like kinase 4 (PLK4), CDK1 or kinesin family members would further disrupt centrosome clustering, eliminate polyploid cancer cells, and enhance tumor cell apoptosis and antineoplastic effects. This dual approach would enhance the anti-tumor efficacy of CDK2 inhibition, potentially overcoming resistance mechanisms. This provides a promising strategy to eradicate persistent lung cancer cells following CDK2 inhibition. Liliya Tyutyunyk-Massey, Zibo Chen, Xiuxia Liu, Masanori Kawakami, Adam Harned, Yeap Ng, Brian Luke, Sameul C. Okpechi, Blessing Ogunlade, Yair Alfaro, Roberto Weigert, Kedar Narayan, Xi Liu, Ethan Dmitrovsky. CDK2 inhibition produces persistent polyploid cancer cells with a survival advantage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5327.

Abstract Background: TRAIL induces apoptosis in many preclinical cancer models including breast cancers and has been extensively studied as a potential cancer therapeutic. However, its efficacy in clinical trials is limited, suggesting an unknown modulatory mechanism responsible for lack of TRAIL activity in vivo. Here, we describe that TRAIL treatment elicits transcriptional changes in TNBC cells that alter the immune milieu. Method: We performed RNAseq of MDA-MB-231 cells treated with TRAIL for different time points, followed by validation with RT-PCR in various TNBC cells. RNAi, silencing key differentially regulated genes, along with RT-PCR, ELISA and CHIP assays, were used to validate RNAseq findings. Elucidation of the functional relevance of the outcome was supported both in vitro and in vivo by chemotaxis assay, cytotoxicity assay, RNAseq analysis of donor isolated neutrophils and intravital microscopy, CODEX analysis in TNBC xenografts from mice treated with the TRAIL respectively. Results: TRAIL treatment of the TNBC significantly induced expression of several cytokines, such as CXCLs 1, 2, 3, 8,11 and IL6, both in vitro and in vivo which are known to affect neutrophil function. Mechanistically, induction of these cytokines was predominantly mediated by death receptor 5 and caspase-8 protein, but not caspase-8 enzymatic activity. GSEA of the RNAseq data indicated that NFKB pathway was significantly enriched. Concordantly, we confirmed that both canonical NFKB1 and non-canonical NFKB2 pathways were activated by TRAIL in vitro and in vivo. However, the induction of the cytokine mRNAs was primarily dependent on the NFKB2 pathway. Neutrophils isolated from healthy human donors incubated with supernatants from TNBC cells in vitro indicated that TRAIL-induced CXCLs and IL6 significantly increased neutrophil chemotaxis. Additionally, CODEX analysis as well as intravital imaging confirmed that TRAIL treatment increases the number of neutrophils in the tumor. Preincubation of neutrophils with supernatants from TRAIL-treated TNBC significantly inhibited their cytotoxic effect against TNBCs. Further, transcriptome analysis of neutrophils incubated with either TRAIL or supernatant of TRAIL treated TNBC revealed significant enrichment of expression of inflammatory cytokine genes, immune modulating and immune checkpoint genes like PDL1. Functional studies with these neutrophils confirmed their suppressive effect on T cell function as well as the effect of TRAIL on decreased neutrophil apoptosis. Conclusion: Collectively, our study suggests the novel role of TRAIL-induced NFKB2-dependent cytokine production promoting neutrophil chemotaxis and immune suppression. This study implies that alterations in the innate immune system may modulate the effects of TRAIL on TNBC tumors. Citation Format: Manjari Kundu, Yoshimi Endo Greer, Lisa Ann Ridnour, David A Wink, Yeap S. Ng, Roberto Weigert, Stan Lipkowitz. Tumor necrosis factor related apoptosis inducing ligand (TRAIL)-induced cytokine production in TNBC promotes neutrophil chemotaxis and immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 347.

Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step. Our results highlight how the actin cytoskeleton tunes its structure to adapt to dynamic changes in the biophysical properties of membranes.

BackgroundRelapsed head and neck squamous cell carcinoma (HNSCC) unrelated to HPV infection carries a poor prognosis. Novel approaches are needed to improve the clinical outcome and prolong survival in this patient population which has poor long-term responses to immune checkpoint blockade. This study evaluated the chemokine receptors CXCR1 and CXCR2 as potential novel targets for the treatment of HPV-negative HNSCC.MethodsExpression of IL-8, CXCR1, and CXCR2 was investigated in HNSCC tissues and human cell line models. Inhibition of CXCR1/2 with the clinical stage, small molecule inhibitor, SX-682, was evaluated in vitro and in vivo using human xenografts and murine models of HNSCC, both as a monotherapy and in combination with the taxane chemotherapy, docetaxel.ResultsHigh levels of IL-8, CXCR1, and CXCR2 expression were observed in HPV-negative compared to HPV-positive HNSCC tumors or cell lines. Treatment of HPV-negative HNSCC cell lines in vitro with SX-682 sensitized the tumor cells to the cytotoxic activity of docetaxel. In vivo, treatment of HNSCC xenograft models with the combination of SX-682 plus docetaxel led to strong anti-tumor control resulting in tumor cures. This phenomenon was associated with an increase of microRNA-200c and a decreased expression of its target, tubulin beta-3, a protein involved in resistance to microtubule-targeting chemotherapies. In vivo treatment of a murine syngeneic model of HNSCC with SX-682 plus docetaxel led to potent anti-tumor efficacy through a simultaneous decrease in suppressive CXCR2+ polymorphonuclear, myeloid-derived suppressor cells and an increase in cytotoxic CD8+ T cells in the combination therapy treated tumors compared to controls.ConclusionsThis study reports, for the first time, mechanistic findings through which the combination of CXCR1/2 inhibition and docetaxel chemotherapy exhibits synergy in models of HPV-negative HNSCC. These findings provide rationale for the use of this novel combination approach to treat HPV-negative HNSCC patients and for future combination studies of CXCR1/2 inhibition, docetaxel, and immune-based therapies.