Marleen Ansems

Purpose Tumor hypoxia is associated with tumor aggressiveness and therapy resistance. Targeted α therapy (TAT) offers a promising approach to eliminate hypoxic tumor cells, as α-particles induce complex DNA damage independent of oxygen availability. We previously demonstrated the therapeutic efficacy of the carbonic anhydrase IX (CAIX)-targeting radiopharmaceutical [ ²²⁵ Ac]Ac-DOTA-MSC3, in combination with immune checkpoint inhibition (ICI). Here, we investigated the radiobiological and immunomodulatory effects of [ ²²⁵ Ac]Ac-DOTA-MSC3 alone and combined with ICI. Methods B16F10-OVA tumor-bearing mice were treated with CAIX-TAT with or without anti-PD-1/anti-CTLA-4 ICI. Tumor and organ uptake of [ ²²⁵ Ac]Ac-DOTA-MSC3 was determined 5 and 8 days post injection. Radiobiological and immunological markers were quantified in CAIX ⁺ and CAIX ⁻ tumor areas. Results [ ²²⁵ Ac]Ac-DOTA-MSC3 selectively accumulated CAIX-expressing hypoxic regions in B16F10-OVA tumors. CAIX-TAT increased DNA damage (γH2AX) compared with PBS control, with stronger effects in CAIX ⁺ than CAIX ⁻ areas. No increase in apoptosis (caspase-3) was observed within the studied timeframe. Compared with ICI alone, CAIX-TAT with ICI increased cGAS foci and CD45 ⁺ immune cell infiltration in both CAIX ⁺ and CAIX ⁻ areas. The combination therapy also increased CD8 ⁺ T-cell and F4/80 ⁺ macrophage numbers, while PD-L1 ⁺ , FOXP3 ⁺ and Ly6G ⁺ cell numbers were unchanged. Conclusion CAIX-TAT induces DNA damage in hypoxic B16F10-OVA tumors and, when combined with ICI, promotes the infiltration of CD45 ⁺ and CD8 ⁺ immune cells. These findings provide a mechanistic rationale for combining CAIX-TAT with ICI to improve the treatment of hypoxic tumors.

Background: Radiotherapy (RT) is a crucial treatment for head and neck squamous cell carcinoma (HNSCC). Recent pre-clinical studies highlight the potential of combining RT with immunotherapy due to RT-induced activation of the cyclic GMP-AMP-synthase (cGAS)/Stimulator of Interferon (IFN) Genes (STING) pathway, yet the clinical implications remain unclear. Methods: In this study, we utilized a cGAS-mNeongreen knock-in tumor model, human HNSCC cell lines and serial tumor biopsies from HNSCC patients to investigate RT-induced cGAS activation and re-localization. cGAS positive micronuclei and IFN stimulated gene (ISG) induction were quantified. In HNSCC patient biopsies, cGAS positive micronuclei were identified in different cellular compartments and IFN/ISG and NF-κB serum levels were correlated to survival. Differences were tested using paired t-tests, Wilcoxon test, One-way and two-way ANOVA or Kaplan Meier curves. Results: Here we show that cGAS positive foci present in micronuclei increase post-RT in both mouse tumor models (P = 0.0305; t-test) and HNSCC patient biopsies (P = 0.0012; Wilcoxon test), concurrently with higher ISG expression. Notably, cGAS re-localization in HNSCC patient biopsies is most prominent in immune cell-infiltrated tumor regions (P = 0.0061; Wilcoxon test). Enhanced systemic type I IFN inducing activity is observed post-RT (P = 0.0040; mixed-effects analysis) and correlates with improved survival (OS P = 0.0146; log-rank test; DFS P = 0.0256; Gehan-Breslow-Wilcoxon test), particularly in HPV positive patients (OS P = 0.0017; log-rank test; DFS P = 0.043; log-rank test). Conclusions: These findings suggest that cGAS re-localization and systemic IFN responses may serve as biomarkers for RT efficacy, providing a basis for optimizing HNSCC treatment protocols, especially when integrated with immunotherapy.

Saponin-based adjuvants (SBAs) distinguish themselves as vaccine adjuvants by instigating a potent activation of CD8+ T cells. Previously, we discovered SBA’s ability to induce cross-presentation in dendritic cells (DCs) leading to CD8+ T cell activation. Moreover, the MHCII^loCD11b^hi bone marrow-derived DC (BMDC) subset was identified to be the most responsive DC subset to SBA treatment. To further investigate SBA’s mode of action, labeling of SBAs was optimized with the fluorescent dye SP-DiIC₁₈(3). Efficient uptake of SBAs occurs specifically by MHCII^loCD11b^hi BMDCs and bone marrow-derived macrophages (BMDMs) in vitro and cDC2s and macrophages ex vivo. Furthermore, SBAs are primarily taken up by clathrin-mediated endocytosis and uptake induces lipid bodies and antigen translocation to the cytosol in MHCII^loCD11b^hi BMDCs and BMDMs. Importantly, BMDMs treated with SBAs exhibit cross-presentation leading to potent CD8+ T cells activation. Our findings explain the potency of SBAs as vaccine adjuvants and contribute to vaccine development.

Cancer-associated fibroblasts (CAFs) represent a group of genotypically non-malignant stromal cells in the tumor micro-environment (TME) of solid tumors that encompasses up to 80% of the tumor volume. Even though the phenotypic diversity and plasticity of CAFs complicates research, it is well-established that CAFs can affect many aspects of tumor progression, including growth, invasion and therapy resistance. Although anti-tumorigenic properties of CAFs have been reported, the majority of research demonstrates a pro-tumorigenic role for CAFs via (in)direct signaling to cancer cells, immunomodulation and extracellular matrix (ECM) remodeling. Following harsh therapeutic approaches such as radio- and/or chemotherapy, CAFs do not die but rather become senescent. Upon conversion towards senescence, many pro-tumorigenic characteristics of CAFs are preserved or even amplified. Senescent CAFs continue to promote tumor cell therapy resistance, modulate the ECM, stimulate epithelial-to-mesenchymal transition (EMT) and induce immunosuppression. Consequently, CAFs play a significant role in tumor cell survival, relapse and potentially malignant transformation of surviving cancer cells following therapy. Modulating CAF functioning in the TME therefore is a critical area of research. Proposed strategies to enhance therapeutic efficacy include reverting senescent CAFs towards a quiescent phenotype or selectively targeting (non-)senescent CAFs. In this review, we discuss CAF functioning in the TME before and during therapy, with a strong focus on radiotherapy. In the future, CAF functioning in the therapeutic TME should be taken into account when designing treatment plans and new therapeutic approaches.

To study head and neck squamous cell carcinomas (HNSCC) in vitro, a large variety of HNSCC cell lines have been developed. Here, we characterize a panel of 22 HNSCC cell lines, thereby providing a tool for research into tumor-specific treatment options in HNSCC. Both human papillomavirus (HPV) positive and HPV negative tumor cell lines were collected from commercial and collaborative sources. Short tandem repeat profiling was used to confirm or characterize the identity of the cell lines. Targeted sequencing was performed using a standard pathology single molecule Molecular Inversion Probe panel to detect mutations for 23 tumor suppressors and oncogenes. HPV status, p16 status, radiosensitivity data, and hypoxia data are summarized from all cell lines. We detected HPV transcripts in five cell lines, all of which overexpressed p16. One HPV negative cell line was also p16 positive. We detected mutations in KIT (SCCNij185), PIK3CA (SCCNij185), and CDKN2A (UT-SCC-5 and UT-SCC-38). TP53 mutations were the most frequent, occurring in 16/22 cell lines. HPV infection and TP53 mutations were almost mutually exclusive, with the exception of 93-VU-147T. The cell lines exhibited a wide range of sensitivities towards hypoxia and irradiation. Here, we provide a description of a set of frequently used HNSCC cell lines with diverse characteristics as found in HNSCC patients.

In the last decade, organoid technology has developed as a primary research tool in basic biological and clinical research. The reliance on poorly defined animal‐derived extracellular matrix, however, severely limits its application in regenerative and translational medicine. Here, a well‐defined, synthetic biomimetic matrix based on polyisocyanide (PIC) hydrogels that support efficient and reproducible formation of mammary gland organoids (MGOs) in vitro is presented. Only decorated with the adhesive peptide RGD for cell binding, PIC hydrogels allow MGO formation from mammary fragments or from purified single mammary epithelial cells. The cystic organoids maintain their capacity to branch for over two months, which is a fundamental and complex feature during mammary gland development. It is found that small variations in the 3D matrix give rise to large changes in the MGO: the ratio of the main cell types in the MGO is controlled by the cell–gel interactions via the cell binding peptide density, whereas gel stiffness controls colony formation efficiency, which is indicative of the progenitor density. Simple hydrogel modifications will allow for future introduction and customization of new biophysical and biochemical parameters, making the PIC platform an ideal matrix for in depth studies into organ development and for application in disease models.