Diffuse reflectance spectroscopy (DRS) is a powerful tool for quantifying optical and physiological tissue properties such as hemoglobin oxygen saturation and vascularity. DRS is increasingly used clinically for distinguishing cancerous lesions from normal tissue. However, its widespread clinical acceptance is still limited due to uncontrolled probe–tissue interface pressure that influences reproducibility and introduces operator-dependent results. In this clinical study, we assessed and validated a pressure-sensing and automatic self-calibration DRS in patients with suspected head and neck squamous cell carcinoma (HNSCC). The clinical study enrolled nineteen patients undergoing HNSCC surgical biopsy procedures. Patients consented to evaluation of this improved DRS system during surgery. For each patient, we obtained 10 repeated measurements on one tumor site and one distant normal location. Using a Monte Carlo-based model, we extracted the hemoglobin saturation data along with total hemoglobin content and scattering properties. A total of twelve cancer tissue samples from HNSCC patients and fourteen normal tissues were analyzed. A linear mixed effects model tested for significance between repeated measurements and compared tumor versus normal tissue. These results demonstrate that cancerous tissues have a significantly lower hemoglobin saturation compared to normal controls (p < 0.001), which may be reflective of tumor hypoxia. In addition, there were minimal changes over time upon probe placement and repeated measurement, indicating that the pressure-induced changes were minimal and repeated measurements did not differ significantly from the initial value. This study demonstrates the feasibility of conducting optical spectroscopy measurements on intact lesions prior to removal during HNSCC procedures, and established that this probe provides diagnostically-relevant physiologic information that may impact further treatment.

Inflammatory breast cancer (IBC), an understudied and lethal breast cancer, is often misdiagnosed due to its unique presentation of diffuse tumor cell clusters in the skin and dermal lymphatics. Here, we describe a window chamber technique in combination with a novel transgenic mouse model that has red fluorescent lymphatics (ProxTom RFP Nu/Nu) to simulate IBC clinicopathological hallmarks. Various breast cancer cells stably transfected to express green or red fluorescent reporters were transplanted into mice bearing dorsal skinfold window chambers. Intravital fluorescence microscopy and the in vivo imaging system (IVIS) were used to serially quantify local tumor growth, motility, length density of lymph and blood vessels, and degree of tumor cell lymphatic invasion over 0–140 h. This short-term, longitudinal imaging time frame in studying transient or dynamic events of diffuse and collectively migrating tumor cells in the local environment and quantitative analysis of the tumor area, motility, and vessel characteristics can be expanded to investigate other cancer cell types exhibiting lymphovascular invasion, a key step in metastatic dissemination. It was found that these models were able to effectively track tumor cluster migration and dissemination, which is a hallmark of IBC clinically, and was recapitulated in these mouse models.

Despite aggressive multimodal treatment with chemoradiation or surgery and adjuvant radiation therapy (RT) +/- chemotherapy, recurrence rates approach 50% for locally advanced human papillomavirus (HPV)-negative HNSCC. Thus, more effective therapeutics are needed in both the upfront and recurrent settings to improve outcomes for HNSCC patients. Ataxia telangiectasia and Rad 3-related (ATR) is a potential therapeutic target that may enhance RT response. ATR is a DNA damage response protein activated by replication stress and single-strand DNA breaks, such as those caused by RT. We hypothesize that ATR inhibition radiosensitizes tumors by preventing DNA repair and forcing HNSCC cells with unrepaired DNA damage into mitosis, leading to cell death. We evaluated the effects of ATR inhibitor BAY 1895344 (BAY) in preclinical models of HPV-negative HNSCC using murine and human cell lines, including MOC1, MOC2, JHU-012, and FaDu. We confirmed dose-dependent inhibition of ATR signaling by BAY using Western blot analysis of downstream phosphorylated CHK1 in cells treated with RT+vehicle versus RT+BAY. Additionally, dose-dependent increase in yH2AX (DNA damage marker) was observed at 18 hours post-RT in cells treated with serial dilutions of BAY vs vehicle. In clonogenic assays, cells treated with 100nM BAY (4h) and 0-8Gy RT demonstrated radiosensitization compared to vehicle. Cell cycle analysis showed that BAY pre-treatment significantly abrogated RT-induced G2/M cell cycle arrest (p<0.01, unpaired t-test). Cells treated with BAY before RT vs vehicle + RT or BAY alone exhibited increased co-staining for yH2AX and pHH3 (mitosis marker) by flow cytometry and immunofluorescence, indicative of mitotic catastrophe. Additionally, MOC2 cells treated with BAY alone or BAY + RT exhibited significantly increased micronuclei and multinucleation events at 24 hours compared to vehicle alone (p=0.0009, unpaired t test) or vehicle + RT (p=0.01), respectively. For in vivo evaluation of BAY in a radioresistant syngeneic mouse model, intramuscular MOC2 tumors were induced in the gastrocnemius of C57BL/6J mice. When tumors reached 70-280 mm³, mice were randomized (n=10/group) to receive vehicle, BAY (40mg/kg), RT (8Gy x 3), or BAY + RT. Image-guided RT was administered every 4 days. Vehicle or BAY was given orally twice on the day of RT or sham RT (morning/evening) and once the morning after RT or sham RT. Median survival was significantly increased in treated groups: 39.5d for BAY + RT vs. 14d for vehicle + sham RT (p=0.0002, Log-rank test), 22d for BAY + sham RT (p=0.0003), and 23d for vehicle + RT (p=0.0007). Thus, the small molecule ATR inhibitor BAY 1895344 radiosensitizes HNSCC in vitro and increases survival combined with RT in vivo, indicating therapeutic potential for targeting ATR in head and neck cancer. The combination of BAY 1895344 (elimusertib), pembrolizumab, and stereotactic body radiation therapy is currently being tested in a phase I clinical trial for patients with unresectable or metastatic HNSCC within a previously irradiated field (NCT04576091). Citation Format: Allison N. Pittman, Diana A. Odhiambo, Rico J. Castillo, Ashlyn G. Rickard, Joshua Chen, Joshua E. Himes, Alex M. Bassil, Nerissa T. Williams, David G. Kirsch, Yvonne M. Mowery. Evaluating the therapeutic potential of ATR inhibitor BAY 1895344 and radiation therapy for head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr IA04.

Background: Present line indications for pembrolizumab are for patients with metastatic or unresectable recurrent (i.e. incurable) head and neck squamous cell carcinoma (HNSCC). However, only a minority of patients on immunotherapy will realize a durable survival benefit because >80% of patients with metastatic HNSCC do not respond to PD-1 blockade. Tumor microenvironment mesenchymal stem cells (MSCs) and cancer associated fibroblasts (CAFs) have been reported significantly contribute to chemotherapy and radiation resistance. Moreover, MSCs have been shown to contribute to an immunosuppressive tumor microenvironment by upregulating PD-L1 in breast cancer models. We have previously shown crenolanib improves MSC mediated cisplatin resistance in vitro through modulation of MSC-mediated activation of AKT signaling, therefore we hypothesize that targeting MSCs may be of therapeutic benefit alone and in combination with anti-PD1 immunotherapy. Methods: Oral cancer was induced in the buccal space of C56/BL6 mice with the murine oral cancer cell lines MOC1 or PD-1 resistant cell line, MOC2. When tumors reached approximately 5 × 5 mm, mice were treated with 15 mg/kg (low dose) or 30 mg/kg crenolanib (high dose) for five consecutive days over 3 weeks. Tissue were harvested for immunohistochemistry and immunofluorescence analysis and ex vivo cell cultures prepared for analysis by western immunoblotting and flow cytometry. Results: There was a significant reduction in tumor volume in mice bearing MOC1 and MOC2 tumors (p<0.03) treated with either low or high dose crenolanib compared to vehicle control. Overall survival was also significantly improved in mice bearing MOC1 tumors treated with high dose crenolanib compared to mice treated with vehicle control (p<0.04) and approached significance in MOC2 mice treated with low dose crenolanib. Tumor sections were imaged by immunofluorescence microscopy. There was a decrease in expression of PDGFR-α on MOC1 tumor cells and α-SMA on tumor microenvironment stromal cells in mice treated with crenolanib compared to vehicle control, suggesting crenolanib targets both cell types. There was a significant reduction in tumor volume (p<0.0001) and improved overall survival (p<0.0004) in mice bearing MOC2 tumors treated with combination crenolanib plus pembrolizumab compared to vehicle plus pembrolizumab. Conclusions: Preliminary in vivo data suggests crenolanib may be efficacious when used in combination with anti-PD1 immunotherapy by inhibiting the immunosuppressive effects of tumor microenvironment MSCs. Citation Format: Xiangfeng Shen, Katherine Gonzalez, Rico Castillo, Ashlyn Rickard, Yvonne Mowery, Tammara Watts. Crenolanib improves PD-1 response and overall survival in immune checkpoint inhibitor resistant murine models of oral squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-093.