Combining immunotherapy and radiation therapy in gastrointestinal cancers: A review.

INTRODUCTION AND PURPOSE: With a significant global impact, treatment of gastrointestinal (GI) cancers still presents with challenges, despite current multimodality approaches in advanced stages. Clinical trials are expanding for checkpoint inhibition (ICI) combined with radiation therapy (RT). This review intends to offer a comprehensive image of the current data regarding the effectiveness of this association, and to reflect on possible directions to further optimize the results. RESULTS: Several early phase studies demonstrated encouraging potential. However, translating preclinical outcomes to clinical settings proves challenging, especially in immunologically "cold" environments. GI cancers exhibit heterogeneity, requiring tailored approaches based on disease stage and patient characteristics. Current results, though promising, lack the power of evidence to influence the general practice. CONCLUSIONS: Finding biomarkers for identifying or converting resistant cancers is essential for maximizing responses, moreover in this context strategic RT parameters need to be carefully considered. Our review emphasizes the significance of having a thorough grasp of how immunology, tumour biology, and treatment settings interact in order to propose novel research avenues and efficient GI cancer therapy.

Characterizing OXPHOS inhibitor-mediated alleviation of hypoxia using high-throughput live cell-imaging.

BACKGROUND: Hypoxia is a common feature of many solid tumors and causes radiotherapy and immunotherapy resistance. Pharmacological inhibition of oxidative phosphorylation (OXPHOS) has emerged as a therapeutic strategy to reduce hypoxia. However, the OXPHOS inhibitors tested in clinical trials caused only moderate responses in hypoxia alleviation or trials were terminated due to dose-limiting toxicities. To improve the therapeutic benefit, FDA approved OXPHOS inhibitors (e.g. atovaquone) were conjugated to triphenylphosphonium (TPP+) to preferentially target cancer cell's mitochondria. In this study, we evaluated the hypoxia reducing effects of several mitochondria-targeted OXPHOS inhibitors and compared them to non-mitochondria-targeted OXPHOS inhibitors using newly developed spheroid models for diffusion-limited hypoxia. METHODS: B16OVA murine melanoma cells and MC38 murine colon cancer cells expressing a HIF-Responsive Element (HRE)-induced Green Fluorescent Protein (GFP) with an oxygen-dependent degradation domain (HRE-eGFP-ODD) were generated to assess diffusion-limited hypoxia dynamics in spheroids. Spheroids were treated with IACS-010759, atovaquone, metformin, tamoxifen or with mitochondria-targeted atovaquone (Mito-ATO), PEGylated mitochondria-targeted atovaquone (Mito-PEG-ATO) or mitochondria-targeted tamoxifen (MitoTam). Hypoxia dynamics were followed and quantified over time using the IncuCyte Zoom Live Cell-Imaging system. RESULTS: Hypoxic cores developed in B16OVA.HRE and MC38.HRE spheroids within 24 h hours after seeding. Treatment with IACS-010759, metformin, atovaquone, Mito-PEG-ATO and MitoTam showed a dose-dependent reduction of hypoxia in both B16OVA.HRE and MC38.HRE spheroids. Mito-ATO only alleviated hypoxia in MC38.HRE spheroids while tamoxifen was not able to reduce hypoxia in any of the spheroid models. The mitochondria-targeted OXPHOS inhibitors demonstrated stronger anti-hypoxic effects compared to the non-mito-targeted OXPHOS inhibitors. CONCLUSIONS: We successfully developed a high-throughput spheroid model in which hypoxia dynamics can be quantified over time. Using this model, we showed that the mitochondria-targeted OXPHOS inhibitors Mito-ATO, Mito-PEG-ATO and MitoTam reduce hypoxia in tumor cells in a dose-dependent manner, potentially sensitizing hypoxic tumor cells for radiotherapy.

Reference intervals for serum 11-oxygenated androgens in children.

OBJECTIVE: Classic androgens such as dehydroepiandrosterone, androstenedione, and testosterone are generally measured for diagnosis and treatment monitoring in children and adolescents with hyperandrogenism, as can occur in congenital adrenal hyperplasia, premature pubarche, or polycystic ovarian syndrome. However, adrenally-derived 11-oxygenated androgens also contribute to the androgen pool and should therefore be considered in clinical management. Nevertheless, paediatric reference intervals are lacking. Therefore, we developed a serum assay to establish reference intervals for four 11-oxygenated androgens in addition to four classic androgens. DESIGN: Reference interval study for serum 11-oxygenated androgens in children. METHODS: We developed and validated a sensitive LC-MS/MS assay and quantified eight serum androgens, including four 11-oxygenated androgens, in serum of 256 healthy children (aged 0-17 years old). An age-dependency for all androgens was observed, and therefore we divided the cohort based on age (prepubertal (n=133; 94 boys, 39 girls) and pubertal (n=123; 52 boys, 71 girls)) to compute reference intervals (2.5th - 97.5th percentiles). RESULTS: In the prepubertal group, there was no significant sex-difference for any of the measured androgens. In the pubertal group, androstenedione, testosterone, and dihydrotestosterone showed a significant difference between boys and girls. In contrast, adrenal androgens dehydroepiandrosterone, 11-hydroxyandrostenedione, 11-ketoandrostenedione, 11-hydroxytestosterone, and 11-ketotestosterone did not. CONCLUSIONS: We developed and validated an assay for 11-oxygenated androgens, in addition to four classic androgens and established reference intervals. This enables a comprehensive evaluation of serum androgen status in children with clinical symptoms of hyperandrogenism.

Evaluation of Ex Vivo Adrenocorticotropic Hormone Responsiveness of Human Fetal Testis.

Testicular adrenal rest tumors (TARTs), commonly occurring in males with congenital adrenal hyperplasia, may arise from chronic stimulation of adrenocorticotropic hormone (ACTH)-sensitive cells in the testes. It is not yet established whether the human fetal testis (HFT) is responsive to ACTH. To investigate this, we cultured HFT tissue with and without ACTH for up to 5 days, and quantified adrenal steroid hormones and expression of adrenal steroidogenic enzymes. Fetal testis and adrenal tissue produced high levels of testosterone and cortisol, respectively, indicating viability. In contrast to fetal adrenal tissues, the expression of ACTH receptor MC2R was either absent or expressed at extremely low levels in ex vivo HFT tissue and no clear response to ACTH in gene expression or steroid hormone production was observed. Altogether, this study suggests that the HFT is unresponsive to ACTH, which would indicate that a TART does not arise from fetal testicular cells chronically exposed to ACTH in utero.

Hormonal control during infancy and testicular adrenal rest tumor development in males with congenital adrenal hyperplasia: a retrospective multicenter cohort study.

IMPORTANCE: Testicular adrenal rest tumors (TARTs), often found in male patients with congenital adrenal hyperplasia (CAH), are benign lesions causing testicular damage and infertility. We hypothesize that chronically elevated adrenocorticotropic hormone exposure during early life may promote TART development. OBJECTIVE: This study aimed to examine the association between commencing adequate glucocorticoid treatment early after birth and TART development. DESIGN AND PARTICIPANTS: This retrospective multicenter (n = 22) open cohort study collected longitudinal clinical and biochemical data of the first 4 years of life using the I-CAH registry and included 188 male patients (median age 13 years; interquartile range: 10-17) with 21-hydroxylase deficiency (n = 181) or 11-hydroxylase deficiency (n = 7). All patients underwent at least 1 testicular ultrasound. RESULTS: TART was detected in 72 (38%) of the patients. Prevalence varied between centers. When adjusted for CAH phenotype, a delayed CAH diagnosis of >1 year, compared with a diagnosis within 1 month of life, was associated with a 2.6 times higher risk of TART diagnosis. TART onset was not predicted by biochemical disease control or bone age advancement in the first 4 years of life, but increased height standard deviation scores at the end of the 4-year study period were associated with a 27% higher risk of TART diagnosis. CONCLUSIONS AND RELEVANCE: A delayed CAH diagnosis of >1 year vs CAH diagnosis within 1 month after birth was associated with a higher risk of TART development, which may be attributed to poor disease control in early life.

Obesity-associated changes in molecular biology of primary breast cancer.

Obesity is associated with an increased risk of developing breast cancer (BC) and worse prognosis in BC patients, yet its impact on BC biology remains understudied in humans. This study investigates how the biology of untreated primary BC differs according to patients' body mass index (BMI) using data from >2,000 patients. We identify several genomic alterations that are differentially prevalent in overweight or obese patients compared to lean patients. We report evidence supporting an ageing accelerating effect of obesity at the genetic level. We show that BMI-associated differences in bulk transcriptomic profile are subtle, while single cell profiling allows detection of more pronounced changes in different cell compartments. These analyses further reveal an elevated and unresolved inflammation of the BC tumor microenvironment associated with obesity, with distinct characteristics contingent on the estrogen receptor status. Collectively, our analyses imply that obesity is associated with an inflammaging-like phenotype. We conclude that patient adiposity may play a significant role in the heterogeneity of BC and should be considered for BC treatment tailoring.

Aberrant APOBEC3B Expression in Breast Cancer Is Linked to Proliferation and Cell Cycle Phase.

APOBEC3B (A3B) is aberrantly overexpressed in a subset of breast cancers, where it associates with advanced disease, poor prognosis, and treatment resistance, yet the causes of A3B dysregulation in breast cancer remain unclear. Here, A3B mRNA and protein expression levels were quantified in different cell lines and breast tumors and related to cell cycle markers using RT-qPCR and multiplex immunofluorescence imaging. The inducibility of A3B expression during the cell cycle was additionally addressed after cell cycle synchronization with multiple methods. First, we found that A3B protein levels within cell lines and tumors are heterogeneous and associate strongly with the proliferation marker Cyclin B1 characteristic of the G2/M phase of the cell cycle. Second, in multiple breast cancer cell lines with high A3B, expression levels were observed to oscillate throughout the cell cycle and again associate with Cyclin B1. Third, induction of A3B expression is potently repressed throughout G0/early G1, likely by RB/E2F pathway effector proteins. Fourth, in cells with low A3B, induction of A3B through the PKC/ncNF-κB pathway occurs predominantly in actively proliferating cells and is largely absent in cells arrested in G0. Altogether, these results support a model in which dysregulated A3B overexpression in breast cancer is the cumulative result of proliferation-associated relief from repression with concomitant pathway activation during the G2/M phase of the cell cycle.

Quantitative Imaging of Hypoxic CAIX-Positive Tumor Areas with Low Immune Cell Infiltration in Syngeneic Mouse Tumor Models.

Limited diffusion of oxygen in combination with increased oxygen consumption leads to chronic hypoxia in most solid malignancies. This scarcity of oxygen is known to induce radioresistance and leads to an immunosuppressive microenvironment. Carbonic anhydrase IX (CAIX) is an enzyme functioning as a catalyzer for acid export in hypoxic cells and is an endogenous biomarker for chronic hypoxia. The aim of this study is to develop a radiolabeled antibody that recognizes murine CAIX to visualize chronic hypoxia in syngeneic tumor models and to study the immune cell population in these hypoxic areas. An anti-mCAIX antibody (MSC3) was conjugated to diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with indium-111 (111In). CAIX expression on murine tumor cells was determined using flow cytometry, and in vitro affinity of [111In]In-MSC3 was analyzed in a competitive binding assay. Ex vivo biodistribution studies were performed to determine in vivo radiotracer distribution. CAIX+ tumor fractions were determined by mCAIX microSPECT/CT, and the tumor microenvironment was analyzed using immunohistochemistry and autoradiography. We showed that [111In]In-MSC3 binds to CAIX-expressing (CAIX+) murine cells in vitro and accumulates in CAIX+ areas in vivo. We optimized the use of [111In]In-MSC3 for preclinical imaging such that it can be applied in syngeneic mouse models and showed that we can quantitatively distinguish between tumor models with varying CAIX+ fractions by ex vivo analyses and in vivo mCAIX microSPECT/CT. Analysis of the tumor microenvironment identified these CAIX+ areas as less infiltrated by immune cells. Together these data demonstrate that mCAIX microSPECT/CT is a sensitive technique to visualize hypoxic CAIX+ tumor areas that exhibit reduced infiltration of immune cells in syngeneic mouse models. In the future, this technique may enable visualization of CAIX expression before or during hypoxia-targeted or hypoxia-reducing treatments. Thereby, it will help optimize immuno- and radiotherapy efficacy in translationally relevant syngeneic mouse tumor models.

Clinical Implications of APOBEC3-Mediated Mutagenesis in Breast Cancer.

Over recent years, members of the APOBEC3 family of cytosine deaminases have been implicated in increased cancer genome mutagenesis, thereby contributing to intratumor and intertumor genomic heterogeneity and therapy resistance in, among others, breast cancer. Understanding the available methods for clinical detection of these enzymes, the conditions required for their (dysregulated) expression, the clinical impact they have, and the clinical implications they may offer is crucial in understanding the current impact of APOBEC3-mediated mutagenesis in breast cancer. Here, we provide a comprehensive review of recent developments in the detection of APOBEC3-mediated mutagenesis and responsible APOBEC3 enzymes, summarize the pathways that control their expression, and explore the clinical ramifications and opportunities they pose. We propose that APOBEC3-mediated mutagenesis can function as a helpful predictive biomarker in several standard-of-care breast cancer treatment plans and may be a novel target for treatment.

Challenges in treatment of patients with non-classic congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) due to 21α-hydroxylase deficiency (21OHD) or 11ß-hydroxylase deficiency (11OHD) are congenital conditions with affected adrenal steroidogenesis. Patients with classic 21OHD and 11OHD have a (nearly) complete enzyme deficiency resulting in impaired cortisol synthesis. Elevated precursor steroids are shunted into the unaffected adrenal androgen synthesis pathway leading to elevated adrenal androgen concentrations in these patients. Classic patients are treated with glucocorticoid substitution to compensate for the low cortisol levels and to decrease elevated adrenal androgens levels via negative feedback on the pituitary gland. On the contrary, non-classic CAH (NCCAH) patients have more residual enzymatic activity and do generally not suffer from clinically relevant glucocorticoid deficiency. However, these patients may develop symptoms due to elevated adrenal androgen levels, which are most often less elevated compared to classic patients. Although glucocorticoid treatment can lower adrenal androgen production, the supraphysiological dosages also may have a negative impact on the cardiovascular system and bone health. Therefore, the benefit of glucocorticoid treatment is questionable. An individualized treatment plan is desirable as patients can present with various symptoms or may be asymptomatic. In this review, we discuss the advantages and disadvantages of different treatment options used in patients with NCCAH due to 21OHD and 11OHD.

Transcriptional comparison of testicular adrenal rest tumors with fetal and adult tissues.

Background: Testicular adrenal rest tumors (TART) are a common complication of unknown cellular origin in patients with congenital adrenal hyperplasia (CAH). These benign tumors have both adrenal and testicular characteristics and are hypothesized to either derive from cells of adrenal origin from the fetal adrenogonadal primordium or by atypical differentiation of adult Leydig-progenitor cells. Objective: This study aims to unravel the identity and etiology of TART. Methods: Co-expression of adrenal-specific CYP11B1 and Leydig cell-specific HSD17B3 in TART was studied using immunohistochemistry. We studied the possibility of TART being derived from atypical differentiation of adult Leydig-progenitor cells by the quantification of adrenal-specific enzyme expression upon adrenocorticotrophic hormone (ACTH)-like stimulation of ex vivo cultured platelet-derived growth factor receptor alpha-positive cells. By comparing the transcriptome of TART (n = 16) with the transcriptome of fetal adrenal (n = 13), fetal testis (n = 5), adult adrenal (n = 11), and adult testis (n = 10) tissues, we explored the identity of TART. Results: We demonstrate co-expression of adrenal-specific CYP11B1 and testis-specific HSD17B3 in TART cells, indicating the existence of a distinct TART cell exhibiting both adrenal and testicular characteristics. Ex vivo cultured adult Leydig-progenitor cells did not express the ACTH-receptor MC2R but did express CYP11B1 upon stimulation. Unsupervised clustering of transcriptome data showed that TART was most similar to adult adrenal tissue, followed by adult testis tissue, and least similar to either fetal tissue. Conclusion: Our data suggest that TART is induced - most likely via activation of a cAMP/protein kinase A-dependent receptor - from a progenitor cell into a unique mature adrenal-like cell type, sometimes exhibiting both adrenal and testicular features.

Low lamin A levels enhance confined cell migration and metastatic capacity in breast cancer.

Aberrations in nuclear size and shape are commonly used to identify cancerous tissue. However, it remains unclear whether the disturbed nuclear structure directly contributes to the cancer pathology or is merely a consequence of other events occurring during tumorigenesis. Here, we show that highly invasive and proliferative breast cancer cells frequently exhibit Akt-driven lower expression of the nuclear envelope proteins lamin A/C, leading to increased nuclear deformability that permits enhanced cell migration through confined environments that mimic interstitial spaces encountered during metastasis. Importantly, increasing lamin A/C expression in highly invasive breast cancer cells reflected gene expression changes characteristic of human breast tumors with higher LMNA expression, and specifically affected pathways related to cell-ECM interactions, cell metabolism, and PI3K/Akt signaling. Further supporting an important role of lamins in breast cancer metastasis, analysis of lamin levels in human breast tumors revealed a significant association between lower lamin A levels, Akt signaling, and decreased disease-free survival. These findings suggest that downregulation of lamin A/C in breast cancer cells may influence both cellular physical properties and biochemical signaling to promote metastatic progression.

Diurnal salivary androstenedione and 17-hydroxyprogesterone levels in healthy volunteers for monitoring treatment efficacy of patients with congenital adrenal hyperplasia.

OBJECTIVE: Treatment of congenital adrenal hyperplasia (CAH) patients with glucocorticoids is often challenging since there is a delicate balance between over- and undertreatment. Treatment can be monitored noninvasively by measuring salivary androstenedione (A4) and 17-hydroxyprogesterone (17-OHP). Optimal treatment monitoring requires the establishment of reference values in saliva. DESIGN: A descriptive study. PATIENTS: For this study saliva of 255 healthy paediatric and adult volunteers with an age range of 4-75 years old was used. MEASUREMENTS: We developed a sensitive liquid chromatography-tandem mass spectrometry method, assessed salivary A4 and 17-OHP stability, and measured A4 and 17-OHP concentrations in saliva collected in the morning, afternoon, and evening. RESULTS: We quantified A4 and 17-OHP concentrations in the morning, afternoon, and evening and demonstrated that there is a significant rhythm with the highest levels in the morning and decreasing levels over the day. A4 and 17-OHP concentrations display an age-dependent pattern. These steroids remain stable in saliva at ambient temperature for up to 5 days. CONCLUSIONS: Good stability of the steroids in saliva enables saliva collection by the patient at home. Since salivary A4 and 17-OHP display a diurnal rhythm and age-dependent pattern, we established reference values for both children and adults at three time points during the day. These reference values support treatment monitoring of children and adults with CAH.

Production of 11-Oxygenated Androgens by Testicular Adrenal Rest Tumors.

CONTEXT: Testicular adrenal rest tumors (TART) are a common complication in males with classic 21-hydroxylase deficiency (21OHD). TART are likely to contribute to the androgen excess in 21OHD patients, but a direct quantification of steroidogenesis from these tumors has not been yet done. OBJECTIVE: We aimed to define the production of 11-oxygenated 19-carbon (11oxC19) steroids by TART. METHODS: Using liquid chromatography-tandem mass spectrometry, steroids were measured in left (n = 7) and right (n = 4) spermatic vein and simultaneously drawn peripheral blood (n = 7) samples from 7 men with 21OHD and TART. For comparison, we also measured the peripheral steroid concentrations in 5 adrenalectomized patients and 12 age- and BMI-matched controls. Additionally, steroids were quantified in TART cell- and adrenal cell-conditioned medium, with and without adrenocorticotropic hormone (ACTH) stimulation. RESULTS: Compared with peripheral blood from 21OHD patients with TART, the spermatic vein samples displayed the highest gradient for 11ß-hydroxytestosterone (11OHT; 96-fold) of the 11oxC19 steroids, followed by 11-ketotestosterone (47-fold) and 11ß-hydroxyandrostenedione (11OHA4; 29-fold), suggesting production of these steroids in TART. TART cells produced higher levels of testosterone and lower levels of A4 and 11OHA4 after ACTH stimulation compared with adrenal cells, indicating ACTH-induced production of testosterone in TART. CONCLUSION: In patients with 21OHD, TART produce 11oxC19 steroids, but in different proportions than the adrenals. The very high ratio of 11OHT in spermatic vs peripheral vein blood suggests the 11-hydroxylation of testosterone by TART, and the in vitro results indicate that this metabolism is ACTH-sensitive.

Optimizing the Timing of Highest Hydrocortisone Dose in Children and Adolescents With 21-Hydroxylase Deficiency.

CONTEXT: Hydrocortisone treatment of young patients with 21-hydroxylase deficiency (21OHD) is given thrice daily, but there is debate about the optimal timing of the highest hydrocortisone dose, either mimicking the physiological diurnal rhythm (morning), or optimally suppressing androgen activity (evening). OBJECTIVE: We aimed to compare 2 standard hydrocortisone timing strategies, either highest dosage in the morning or evening, with respect to hormonal status throughout the day, nocturnal blood pressure (BP), and sleep and activity scores. METHODS: This 6-week crossover study included 39 patients (aged 4-19 years) with 21OHD. Patients were treated for 3 weeks with the highest hydrocortisone dose in the morning, followed by 3 weeks with the highest dose in the evening (n = 21), or vice versa (n = 18). Androstenedione (A4) and 17-hydroxyprogesterone (17OHP) levels were quantified in saliva collected at 5 am; 7 am; 3 pm; and 11 pm during the last 2 days of each treatment period. The main outcome measure was comparison of saliva 17OHP and A4 levels between the 2 treatment strategies. RESULTS: Administration of the highest dose in the evening resulted in significantly lower 17OHP levels at 5 am, whereas the highest dose in the morning resulted in significantly lower 17OHP and A4 levels in the afternoon. The 2 treatment dose regimens were comparable with respect to averaged daily hormone levels, nocturnal BP, and activity and sleep scores. CONCLUSION: No clear benefit for either treatment schedule was established. Given the variation in individual responses, we recommend individually optimizing dose distribution and monitoring disease control at multiple time points.

Quality of Life in Men With Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is a disorder of adrenal steroid biosynthesis, leading to hypocortisolism, hypoaldosteronism, and hyperandrogenism. Impaired quality of life (QoL) has been demonstrated in women with CAH, but data on men with CAH are scarce. We hypothesized that disease severity and poor treatment control are inversely associated with QoL. In this study, 109 men (16-68 years) with 21OHD were included. The WHOQOL-BREF questionnaire was used to measure self-reported QoL domain scores on a 0-100 scale, where higher scores reflect better QoL. QoL domain scores were compared to published data on healthy and chronically ill reference populations from France, Germany, the Netherlands, and the United Kingdom. Differences in QoL scores among groups of disease severity and treatment control were tested within the study population. Overall, the men with CAH in this study appeared to rate their QoL as good. Median domain scores were 78.6 (IQR: 67.9-85.7) for physical health, 79.2 (IQR: 66.7-87.5) for psychological health, 75.0 (IQR: 58.3-83.3) for social relationships, and 81.3 (IQR: 71.9-90.6) for environment. In general, these scores were similar to WHOQOL-BREF domain scores in healthy references and higher compared to chronically ill reference populations. The domain scores did not differ among genotype groups, but patients with undertreatment or increased 17-hydroxyprogestrone concentrations scored higher on several QoL domains (p<0.05). Patients treated with dexamethasone or prednisone scored higher on the physical health, psychological health, and social relationships domains, but not on the environmental domain. In conclusion, QoL domain scores appeared to be comparable to healthy reference populations and higher compared to patients with a chronic illness. QoL was not influenced by genotype, but undertreatment and use of dexamethasone or prednisone were associated with higher QoL.

Genotyping and Characterization of HPV Status, Hypoxia, and Radiosensitivity in 22 Head and Neck Cancer Cell Lines.

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.

Improving Breast Cancer Treatment Specificity Using Aptamers Obtained by 3D Cell-SELEX.

Three-dimensional spheroids of non-malignant MCF10A and malignant SKBR3 breast cells were used for subsequent 3D Cell-SELEX to generate aptamers for specific binding and treatment of breast cancer cells. Using 3D Cell-SELEX combined with Next-Generation Sequencing and bioinformatics, ten abundant aptamer families with specific structures were identified that selectively bind to SKBR3, and not to MCF10A cells. Multivalent aptamer polymers were synthesized by co-polymerization and analyzed for binding performance as well as therapeutic efficacy. Binding performance was determined by confocal fluorescence imaging and revealed specific binding and efficient internalization of aptamer polymers into SKBR3 spheroids. For therapeutic purposes, DNA sequences that intercalate the cytotoxic drug doxorubicin were co-polymerized into the aptamer polymers. Viability tests show that the drug-loaded polymers are specific and effective in killing SKBR3 breast cancer cells. Thus, the 3D-selected aptamers enhanced the specificity of doxorubicin against malignant over non-malignant breast cells. The innovative modular DNA aptamer platform based on 3D Cell SELEX and polymer multivalency holds great promise for diagnostics and treatment of breast cancer.

Letter to the editor: Hypoxia kinetics and histology in combined radiotherapy and oxidative phosphorylation inhibition effects on antitumor immunity.

In response to the recent paper by Chen et al investigating the triple combination of oxidative phosphorylation inhibition, immunotherapy and radiotherapy, we would like to stress that after irradiation, a strong reduction in hypoxia (within 24 hours) can be followed by a strong increase (several days). This is especially the case with larger fraction sizes of radiation therapy, which are often applied in combination with immunotherapy, and is likely to be tumor dependent. All together this may strongly affect the synergistic effect of such a triple combination therapy.

Radiotherapy and cGAS/STING signaling: Impact on MDSCs in the tumor microenvironment.

Myeloid derived suppressor cells (MDSCs) are a highly heterogeneous population of immature immune cells with immunosuppressive functions that are recruited to the tumor microenvironment (TME). MDSCs promote tumor growth and progression by inhibiting immune effector cell proliferation and function. MDSCs are affected by both novel anti-cancer therapies targeting the immune system to promote anti-tumor immunity, as well as by conventional treatments such as radiotherapy. Following radiotherapy, cytoplasmic double stranded DNA stimulates the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway, resulting in type I interferon production. Effectiveness of radiotherapy and cGAS/STING signaling are closely intertwined: activation of cGAS and STING is key to generate systemic anti-tumor immunity after irradiation. This review focuses on how radiotherapy and cGAS/STING signaling in MDSCs and/or tumor cells impact MDSC recruitment, expansion and function. The influence of conventional and ablative radiotherapy treatment schedules, inflammatory response following radiotherapy, and hypoxia are discussed as MDSC modulators.