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Background: Recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) generally has a poor prognosis for patients with limited treatment options. While incorporating immune checkpoint inhibitors (ICIs) has now become the standard of care, the efficacy is variable, with only a subset of patients responding. The complexity of the tumor microenvironment (TME) and the role of tertiary lymphoid structures (TLS) have emerged as critical determinants for immunotherapeutic response., Methods: In this study, we analyzed two independently collected R/M HNSCC patient tissue cohorts to better understand the role of TLS in response to ICIs. Utilizing a multi-omics approach, we first performed targeted proteomic profiling using the Nanostring GeoMx Digital Spatial Profiler to quantify immune-related protein expression with spatial resolution. This was further characterized by spatially resolved whole transcriptome profiling of TLSs and germinal centers (GCs). Deeper single-cell resolved proteomic profiling of the TLSs was performed using the Akoya Biosciences Phenocycler Fusion platform., Results: Our proteomic analysis revealed the presence of T lymphocyte markers, including CD3, CD45, and CD8, expressing cells and upregulation of immune checkpoint marker PD-L1 within tumor compartments of patients responsive to ICIs, indicative of 'hot tumor' phenotypes. We also observed the presence of antigen-presenting cells marked by expression of CD40, CD68, CD11c, and CD163 with upregulation of antigen-presentation marker HLA-DR, in patients responding to ICIs. Transcriptome analysis of TLS and GCs uncovered a marked elevation in the expression of genes related to immune modulation, diverse immune cell recruitment, and a potent interferon response within the TLS structure. Notably, the distribution of TLS-tumor distance was found to be significantly different across response groups (H = 9.28, p = 0.026). The proximity of TLSs to tumor cells was found to be a critical indicator of ICI response, implying that patients with TLSs located further from tumor cells have worse outcomes., Conclusion: The study underscores the multifaceted role of TLSs in modulating the immunogenic landscape of the TME in R/M HNSCC, likely influencing the efficacy of ICIs. Spatially resolved multi-omics approaches offer valuable insights into potential biomarkers for ICI response and highlight the importance of profiling the TME complexity when developing therapeutic strategies and patient stratification., (© 2024. The Author(s).)

Background: Triple-negative breast cancer (TNBC) is a sub-classification of breast carcinomas, which leads to poor survival outcomes for patients. TNBCs do not possess the hormone receptors that are frequently targeted as a therapeutic in other cancer subtypes and, therefore, chemotherapy remains the standard treatment for TNBC. Nuclear envelope proteins are frequently dysregulated in cancer cells, supporting their potential as novel cancer therapy targets. The Lem-domain (Lem-D) (LAP2, Emerin, MAN1 domain, and Lem-D) proteins are a family of inner nuclear membrane proteins, which share a ~45-residue Lem-D. The Lem-D proteins, including Ankle2, Lemd2, TMPO, and Emerin, have been shown to be associated with many of the hallmarks of cancer. This study aimed to define the association between the Lem-D proteins and TNBC and determine whether these proteins could be promising therapeutic targets., Methods: GENT2, TCGA, and KM plotter were utilized to investigate the expression and prognostic implications of several Lem-D proteins: Ankle2, TMPO, Emerin, and Lemd2 in publicly available breast cancer patient data. Immunoblotting and immunofluorescent analysis of immortalized non-cancerous breast cells and a panel of TNBC cells were utilized to establish whether protein expression of the Lem-D proteins was significantly altered in TNBC. SiRNA was used to decrease individual Lem-D protein expression, and functional assays, including proliferation assays and apoptosis assays, were conducted., Results: The Lem-D proteins were generally overexpressed in TNBC patient samples at the mRNA level and showed variable expression at the protein level in TNBC cell lysates. Similarly, protein levels were generally negatively correlated with patient survival outcomes. siRNA-mediated depletion of the individual Lem-D proteins in TNBC cells induced aberrant nuclear morphology, decreased proliferation, and induced cell death. However, minimal effects on nuclear morphology or cell viability were observed following Lem-D depletion in non-cancerous MCF10A cells., Conclusion: There is evidence to suggest that Ankle2, TMPO, Emerin, and Lemd2 expressions are correlated with breast cancer patient outcomes, but larger patient sample numbers are required to confirm this. siRNA-mediated depletion of these proteins was shown to specifically impair TNBC cell growth, suggesting that the Lem-D proteins may be a specific anti-cancer target., Competing Interests: Authors EB, DR, and KO are founders of Carpe Vitae Pharmaceuticals. EB, KO, and DR are inventors on provisional patent applications filed by Queensland University of Technology. The remaining 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 © 2024 Rose, Burgess, Cheong, Adams, Shahrouzi, O’Byrne, Richard and Bolderson.)

Background: 5-Fluorouracil (5-FU) remains a core component of systemic therapy for colorectal cancer (CRC). However, response rates remain low, and development of therapy resistance is a primary issue. Combinatorial strategies employing a second agent to augment the therapeutic effect of chemotherapy is predicted to reduce the incidence of treatment resistance and increase the durability of response to therapy., Methods: Here, we employed quantitative proteomics approaches to identify novel druggable proteins and molecular pathways that are deregulated in response to 5-FU, which might serve as targets to improve sensitivity to chemotherapy. Drug combinations were evaluated using 2D and 3D CRC cell line models and an ex vivo culture model of a patient-derived tumour., Results: Quantitative proteomics identified upregulation of the mitosis-associated protein Aurora B (AURKB), within a network of upregulated proteins, in response to a 24 h 5-FU treatment. In CRC cell lines, AURKB inhibition with the dihydrogen phosphate prodrug AZD1152, markedly improved the potency of 5-FU in 2D and 3D in vitro CRC models. Sequential treatment with 5-FU then AZD1152 also enhanced the response of a patient-derived CRC cells to 5-FU in ex vivo cultures., Conclusions: AURKB inhibition may be a rational approach to augment the effectiveness of 5-FU chemotherapy in CRC., (© 2024. The Author(s).)

Human single-stranded DNA binding protein 1 (hSSB1) forms a heterotrimeric complex, known as a sensor of single-stranded DNA binding protein 1 (SOSS1), in conjunction with integrator complex subunit 3 (INTS3) and C9ORF80. This sensory protein plays an important role in homologous recombination repair of double-strand breaks in DNA to efficiently recruit other repair proteins at the damaged sites. Previous studies have identified elevated hSSB1-mediated DNA repair activities in various cancers, highlighting its potential as an anticancer target. While prior efforts have focused on inhibiting hSSB1 by targeting its DNA binding domain, this study seeks to explore the inhibition of the hSSB1 function by disrupting its interaction with the key partner protein INTS3 in the SOSS1 complex. The investigative strategy entails a molecular docking-based screening of a specific compound library against the three-dimensional structure of INTS3 at the hSSB1 binding interface. Subsequent assessments involve in vitro analyses of protein-protein interaction (PPI) disruption and cellular effects through co-immunoprecipitation and immunofluorescence assays, respectively. Moreover, the study includes an evaluation of the structural stability of ligands at the INTS3 hot-spot site using molecular dynamics simulations. The results indicate a potential in vitro disruption of the INTS3-hSSB1 interaction by three of the tested compounds obtained from the virtual screening with one impacting the recruitment of hSSB1 and INTS3 to chromatin following DNA damage. To our knowledge, our results identify the first set of drug-like compounds that functionally target INTS3-hSSB1 interaction, and this provides the basis for further biophysical investigations that should help to speed up PPI inhibitor discovery., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Objective: The recent availability of standing computed tomography (CT) for equine paranasal sinus disease has enhanced diagnosis and enabled more targeted surgery compared to radiography and sinoscopy. To date, there have been no studies which have critically evaluated the benefit of preoperative CT versus radiography and sinoscopy on complications and outcome in horses undergoing sinus trephination., Study Design: Retrospective study., Animals: A total of 229 equids., Methods: The medical records of equids (229) presented for sinusitis treated via trephination that had CT, radiography and/or sinoscopy at time of surgery between 2009 and 2022 were reviewed. Outcome and complications were evaluated for six different pre- and intraoperative imaging modalities., Results: The six groups did not differ in demographics or disease category, though equids with less weight (p = .0179) and shorter disease duration (p = .0075) were more likely to have radiography and sinoscopy based surgical planning. Short-term postoperative complications occurred in 30.1% and were higher in groups using preoperative CT imaging (p = .01), with hemorrhage being the most common surgical complication. Following initial trephination surgery, 57.0% (127/223) of cases resolved and there was no difference between the imaging groups. Final resolution after follow-up medical or surgical treatment increased to 94.6% (211/223) and was not different between the imaging groups or between primary or secondary sinusitis. Additional nasal fenestrations to improve sinonasal drainage, maxillary septal bulla fenestrations and trephinations to treat nasal conchal bullae were made in the CT groups., Clinical Significance: CT provided additional diagnostic information and enabled different surgical approaches but did not improve resolution in this study population., (© 2023 The Authors. Veterinary Surgery published by Wiley Periodicals LLC on behalf of American College of Veterinary Surgeons.)

Human single-stranded DNA binding protein 1 (hSSB1) is critical to preserving genome stability, interacting with single-stranded DNA (ssDNA) through an oligonucleotide/oligosaccharide binding-fold. The depletion of hSSB1 in cell-line models leads to aberrant DNA repair and increased sensitivity to irradiation. hSSB1 is over-expressed in several types of cancers, suggesting that hSSB1 could be a novel therapeutic target in malignant disease. hSSB1 binding studies have focused on DNA; however, despite the availability of 3D structures, small molecules targeting hSSB1 have not been explored. Quinoline derivatives targeting hSSB1 were designed through a virtual fragment-based screening process, synthesizing them using AlphaLISA and EMSA to determine their affinity for hSSB1. In parallel, we further screened a structurally diverse compound library against hSSB1 using the same biochemical assays. Three compounds with nanomolar affinity for hSSB1 were identified, exhibiting cytotoxicity in an osteosarcoma cell line. To our knowledge, this is the first study to identify small molecules that modulate hSSB1 activity. Molecular dynamics simulations indicated that three of the compounds that were tested bound to the ssDNA-binding site of hSSB1, providing a framework for the further elucidation of inhibition mechanisms. These data suggest that small molecules can disrupt the interaction between hSSB1 and ssDNA, and may also affect the ability of cells to repair DNA damage. This test study of small molecules holds the potential to provide insights into fundamental biochemical questions regarding the OB-fold.

Epithelial-mesenchymal plasticity (EMP) is a hallmark of cancer. By enabling cells to shift between different morphological and functional states, EMP promotes invasion, metastasis and therapy resistance. We report that near-diploid non-cancerous human epithelial lung cells spontaneously shift along the EMP spectrum without genetic changes. Strikingly, more than half of single cell-derived clones adopt a mesenchymal morphology. We independently characterise epithelial-like and mesenchymal-like clones. Mesenchymal clones lose epithelial markers, display larger cell aspect ratios and lower motility, with mostly unaltered proliferation rates. Stemness marker expression and metabolic rewiring diverge independently of phenotypes. In 3D culture, more epithelial clones become mesenchymal-like. Thus, non-cancerous epithelial cells may acquire cancer metastasis-associated features prior to genetic alterations and cancerous transformation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The repair of double-strand DNA breaks (DSBs) by homologous recombination is crucial in the maintenance of genome integrity. While the key role of the Mre11-Rad50-Nbs1 (MRN) complex in repair is well known, hSSB1 (SOSSB and OBFC2B), one of the main components of the sensor of single-stranded DNA (SOSS) protein complex, has also been shown to rapidly localize to DSB breaks and promote repair. We have previously demonstrated that hSSB1 binds directly to Nbs1, a component of the MRN complex, in a DNA damage-independent manner. However, recruitment of the MRN complex has also been demonstrated by an interaction between Integrator Complex Subunit 3 (INTS3; also known as SOSSA), another member of the SOSS complex, and Nbs1. In this study, we utilize a combined approach of in silico, biochemical, and functional experiments to uncover the molecular details of INTS3 binding to Nbs1. We demonstrate that the forkhead-associated domain of Nbs1 interacts with INTS3 via phosphorylation-dependent binding to INTS3 at Threonine 592, with contributions from Serine 590. Based on these data, we propose a model of MRN recruitment to a DSB via INTS3., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Glucose metabolism and DNA repair are fundamental cellular processes frequently dysregulated in cancer. In this study, we define a direct role for the glycolytic Aldolase A (ALDOA) protein in DNA double-strand break (DSB) repair. ALDOA is a fructose biphosphate Aldolase that catalyses fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), during glycolysis. Here, we show that upon DNA damage induced by ionising radiation (IR), ALDOA translocates from the cytoplasm into the nucleus, where it partially co-localises with the DNA DSB marker γ-H2AX. DNA damage was shown to be elevated in ALDOA-depleted cells prior to IR and following IR the damage was repaired more slowly. Consistent with this, cells depleted of ALDOA exhibited decreased DNA DSB repair via non-homologous end-joining and homologous recombination. In support of the defective repair observed in its absence, ALDOA was found to associate with the major DSB repair effector kinases, DNA-dependent Protein Kinase (DNA-PK) and Ataxia Telangiectasia Mutated (ATM) and their autophosphorylation was decreased when ALDOA was depleted. Together, these data establish a role for an essential metabolic protein, ALDOA in DNA DSB repair and suggests that targeting ALDOA may enable the concurrent targeting of cancer metabolism and DNA repair to induce tumour cell death., (© 2023. Springer Nature Limited.)

Background: There is an under-representation of Black, Asian and minority ethnic nurses in senior positions within the UK's national healthcare system., Objectives: To understand student nurses' perspectives on the role of race and ethnicity on career expectations, course learning and delivery, and areas for additional training and skill development for all nurses in understanding structural inequalities in healthcare., Design: Qualitative study involving semi-structured interviews., Settings: University in south-east England, UK., Participants: 15 nursing students (14 women, one man) from a range of ethnic backgrounds, age-groups and nationalities., Methods: Interviews lasting 30-60 min were conducted with nursing students and thematic analyses undertaken., Results: Four inter-related themes were constructed: altered career expectations, lack of understanding, absent discussion of racism and missing representation. Experiences of racism were not uncommon for students from Black, Asian and minority ethnic backgrounds and these experiences affected these students' career expectations. Students described a lack of understanding about racism and that it was a taboo topic on their course and in placements., Conclusions: Findings highlight an urgent need for universities to challenge existing nursing curricula to ensure inclusive, anti-racist educational provision that works equitably for all future nurses. The importance of representation was highlighted among those who deliver courses, in the content of nursing curriculum through inclusive education, decolonised curricula and with student voices embedded to enable the development of culturally-competent nursing graduates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

The composition and activation status of the cellular milieu contained within the tumour microenvironment (TME) is becoming increasingly recognized as a driving factor for immunotherapy response. Here, we employed multiplex immunohistochemistry (mIHC), and digital spatial profiling (DSP) to capture the targeted immune proteome and transcriptome of tumour and TME compartments from an immune checkpoint inhibitor (ICI)-treated (n = 41) non-small cell lung cancer (NSCLC) patient cohort. We demonstrate by mIHC that the interaction of CD68 + macrophages with PD1 + , FoxP3 + cells is enriched in ICI refractory tumours (p = 0.012). Patients responsive to ICI therapy expressed higher levels of IL2 receptor alpha (CD25, p = 0.028) within their tumour compartments, which corresponded with increased IL2 mRNA (p = 0.001) within their stroma. In addition, stromal IL2 mRNA levels positively correlated with the expression of pro-apoptotic markers cleaved caspase 9 (p = 2e -5 ) and BAD (p = 5.5e -4 ) and negatively with levels of memory marker, CD45RO (p = 7e -4 ). Immuno-inhibitory markers CTLA-4 (p = 0.021) and IDO-1 (p = 0.023) were suppressed in ICI-responsive patients. Tumour expression of CD44 was depleted in the responsive patients (p = 0.02), while higher stromal expression of one of its ligands, SPP1 (p = 0.008), was observed. Cox survival analysis also indicated tumour CD44 expression was associated with poorer prognosis (hazard ratio [HR] = 1.61, p = 0.01), consistent with its depletion in ICI-responsive patients. Through multi-modal approaches, we have dissected the characteristics of NSCLC immunotherapy treatment groups and provide evidence for the role of several markers including IL2, CD25, CD44 and SPP1 in the efficacy of current generations of ICI therapy., (© 2023 The Authors. Immunology published by John Wiley & Sons Ltd.)

Background: Activation and regulation of androgen receptor (AR) signaling and the DNA damage response impact the prostate cancer (PCa) treatment modalities of androgen deprivation therapy (ADT) and radiotherapy. Here, we have evaluated a role for human single-strand binding protein 1 (hSSB1/NABP2) in modulation of the cellular response to androgens and ionizing radiation (IR). hSSB1 has defined roles in transcription and maintenance of genome stability, yet little is known about this protein in PCa., Methods: We correlated hSSB1 with measures of genomic instability across available PCa cases from The Cancer Genome Atlas (TCGA). Microarray and subsequent pathway and transcription factor enrichment analysis were performed on LNCaP and DU145 prostate cancer cells., Results: Our data demonstrate that hSSB1 expression in PCa correlates with measures of genomic instability including multigene signatures and genomic scars that are reflective of defects in the repair of DNA double-strand breaks via homologous recombination. In response to IR-induced DNA damage, we demonstrate that hSSB1 regulates cellular pathways that control cell cycle progression and the associated checkpoints. In keeping with a role for hSSB1 in transcription, our analysis revealed that hSSB1 negatively modulates p53 and RNA polymerase II transcription in PCa. Of relevance to PCa pathology, our findings highlight a transcriptional role for hSSB1 in regulating the androgen response. We identified that AR function is predicted to be impacted by hSSB1 depletion, whereby this protein is required to modulate AR gene activity in PCa., Conclusions: Our findings point to a key role for hSSB1 in mediating the cellular response to androgen and DNA damage via modulation of transcription. Exploiting hSSB1 in PCa might yield benefits as a strategy to ensure a durable response to ADT and/or radiotherapy and improved patient outcomes., (© 2023 The Authors. The Prostate published by Wiley Periodicals LLC.)

Mucosal head and neck squamous cell carcinoma (HNSCC) are the seventh most common cancer, with approximately 50% of patients living beyond 5 years. Immune checkpoint inhibitors (ICIs) have shown promising results in patients with recurrent or metastatic (R/M) disease, however, only a subset of patients benefit from immunotherapy. Studies have implicated the tumor microenvironment (TME) of HNSCC as a major factor in therapy response, highlighting the need to better understand the TME, particularly by spatially resolved means to determine cellular and molecular components. Here, we employed targeted spatial profiling of proteins on a cohort of pre-treatment tissues from patients with R/M disease to identify novel biomarkers of response within the tumor and stromal margins. By grouping patient outcome categories into response or non-response, based on Response Evaluation Criteria in Solid Tumors (RECIST) we show that immune checkpoint molecules, including PD-L1, B7-H3, and VISTA, were differentially expressed. Patient responders possessed significantly higher tumor expression of PD-L1 and B7-H3, but lower expression of VISTA. Analysis of response subgroups indicated that tumor necrosis factor receptor (TNFR) superfamily members including OX40L, CD27, 4-1BB, CD40, and CD95/Fas, were associated with immunotherapy outcome. CD40 expression was higher in patient-responders than non responders, while CD95/Fas expression was lower in patients with partial response (PR) relative to those with stable disease (SD) and progressive disease (PD). Furthermore, we found that high 4-1BB expression in the tumor compartment, but not in the stroma, was associated with better overall survival (OS) (HR= 0.28, p-adjusted= 0.040). Moreover, high CD40 expression in tumor regions (HR= 0.27, p-adjusted= 0.035), and high CD27 expression in the stroma (HR= 0.2, p-adjusted=0.032) were associated with better survival outcomes. Taken together, this study supports the role of immune checkpoint molecules and implicates the TNFR superfamily as key players in immunotherapy response in our cohort of HNSCC. Validation of these findings in a prospective study is required to determine the robustness of these tissue signatures., Competing Interests: NM, BC, NJ, and OB are employees of Akoya Biosciences. BH, advisory board member of Merck Sharpe and Dohme, Bristol Myers Squibb, Astra Zeneca, Pfizer, Takeda, Sanofi, Eisai. The remaining 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 © 2023 Sadeghirad, Liu, Monkman, Ma, Cheikh, Jhaveri, Tan, Warkiani, Adams, Nguyen, Ladwa, Braubach, O’Byrne, Davis, Hughes and Kulasinghe.)

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase and its cofactor, Cdh1, regulate the expression of several cell-cycle proteins and their functions during mitosis. Levels of the protein cell division cycle-associated protein 3 (CDCA3), which is functionally required for mitotic entry, are regulated by APC/C Cdh1 . CDCA3 is an intrinsically disordered protein and contains both C-terminal KEN box and D-box recognition motifs, enabling binding to Cdh1. Our previous findings demonstrate that CDCA3 has a phosphorylation-dependent non-canonical ABBA-like motif within the linker region bridging these two recognition motifs and is required for efficient binding to Cdh1. Here, we sought to identify and further characterize additional residues that participate within this ABBA-like motif using detailed in vitro experiments and in silico modeling studies. We identified the role of H-bonds, hydrophobic and ionic interactions across the CDCA3 ABBA-like motif in the linker region between KEN and D-box motifs. This linker region adopts a well-defined structure when bound to Cdh1 in the presence of phosphorylation. Upon alanine mutation, the structure of this region is lost, leading to higher flexibility, and alteration in affinities due to binding to alternate sites on Cdh1. Our findings identify roles for the anchoring residues in the non-canonical ABBA-like motif to promote binding to the APC/C Cdh1 and regulation of CDCA3 protein levels., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

3D organoid model technologies have led to the development of innovative tools for cancer precision medicine. Yet, the gold standard culture system (Matrigel®) lacks the ability for extensive biophysical manipulation needed to model various cancer microenvironments and has inherent batch-to-batch variability. Tunable hydrogel matrices provide enhanced capability for drug testing in breast cancer (BCa), by better mimicking key physicochemical characteristics of this disease’s extracellular matrix. Here, we encapsulated patient-derived breast cancer cells in bioprinted polyethylene glycol-derived hydrogels (PEG), functionalized with adhesion peptides (RGD, GFOGER and DYIGSR) and gelatin-derived hydrogels (gelatin methacryloyl; GelMA and thiolated-gelatin crosslinked with PEG-4MAL; GelSH). Within ranges of BCa stiffnesses (1−6 kPa), GelMA, GelSH and PEG-based hydrogels successfully supported the growth and organoid formation of HR+,−/HER2+,− primary cancer cells for at least 2−3 weeks, with superior organoid formation within the GelSH biomaterial (up to 268% growth after 15 days). BCa organoids responded to doxorubicin, EP31670 and paclitaxel treatments with increased IC50 concentrations on organoids compared to 2D cultures, and highest IC50 for organoids in GelSH. Cell viability after doxorubicin treatment (1 µM) remained >2-fold higher in the 3D gels compared to 2D and doxorubicin/paclitaxel (both 5 µM) were ~2.75−3-fold less potent in GelSH compared to PEG hydrogels. The data demonstrate the potential of hydrogel matrices as easy-to-use and effective preclinical tools for therapy assessment in patient-derived breast cancer organoids.

Rationale: An antibody-drug conjugate (ADC) is a targeted therapy consisting of a cytotoxic payload that is linked to an antibody which targets a protein enriched on malignant cells. Multiple ADCs are currently used clinically as anti-cancer agents significantly improving patient survival. Herein, we evaluated the rationale of targeting the cell surface oncoreceptor CUB domain-containing protein 1 (CDCP1) using ADCs and assessed the efficacy of CDCP1-directed ADCs against a range of malignant tumors. Methods: CDCP1 mRNA expression was evaluated using large transcriptomic datasets of normal/tumor samples for 23 types of cancer and 15 other normal organs, and CDCP1 protein expression was examined in 34 normal tissues, >300 samples from six types of cancer, and in 49 cancer cell lines. A recombinant human/mouse chimeric anti-CDCP1 antibody (ch10D7) was labelled with 89 Zirconium or monomethyl auristatin E (MMAE) and tested in multiple pre-clinical cancer models including 36 cancer cell lines and three mouse xenograft models. Results: Analysis of CDCP1 expression indicates elevated CDCP1 expression in the majority of the cancers and restricted expression in normal human tissues. Antibody ch10D7 demonstrates a high affinity and specificity for CDCP1 inducing cell signalling via Src accompanied by rapid internalization of ch10D7/CDCP1 complexes in cancer cells . 89 Zirconium-labelled ch10D7 accumulates in CDCP1 expressing cells enabling detection of pancreatic cancer xenografts in mice by PET imaging. Cytotoxicity of MMAE-labelled ch10D7 against kidney, colorectal, lung, ovarian, pancreatic and prostate cancer cells in vitro , correlates with the level of CDCP1 on the plasma membrane. ch10D7-MMAE displays robust anti-tumor effects against mouse xenograft models of pancreatic, colorectal and ovarian cancer. Conclusion: CDCP1 directed imaging agents will be useful for selecting cancer patients for personalized treatment with cytotoxin-loaded CDCP1 targeting agents including antibody-drug conjugates., Competing Interests: Competing Interests: Thomas Kryza, Simon Puttick, Yaowu He and John D. Hooper are inventors on a patent covering the use of antibody 10D7 used in this study. No potential conflicts of interest were disclosed by the other authors., (© The author(s).)

Head and neck squamous cell carcinoma (HNSCC) represents a heterogeneous group of tumors. While significant progress has been made using multimodal treatment, the 5-year survival remains at 50%. Developing effective therapies, such as immunotherapy, will likely lead to better treatment of primary and metastatic disease. However, not all HNSCC tumors respond to immune checkpoint blockade therapy. Understanding the complex cellular composition and interactions of the tumor microenvironment is likely to lead to new knowledge for effective therapies and treatment resistance. In this review, we discuss HNSCC characteristics, predictive biomarkers, factors influencing immunotherapy response, with a focus on the tumor microenvironment., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that has few effective treatment options due to its lack of targetable hormone receptors. Whilst the degree of tumour infiltrating lymphocytes (TILs) has been shown to associate with therapy response and prognosis, deeper characterization of the molecular diversity that may mediate chemotherapeutic response is lacking. Here we applied targeted proteomic analysis of both chemotherapy sensitive and resistant TNBC tissue samples by the Nanostring GeoMx Digital Spatial Platform (DSP). By quantifying 68 targets in the tumour and tumour microenvironment (TME) compartments and performing differential expression analysis between responsive and non-responsive tumours, we show that increased ER-alpha expression and decreased 4-1BB and MART1 within the stromal compartments is associated with adjuvant chemotherapy response. Similarly, higher expression of GZMA, STING and fibronectin and lower levels of CD80 were associated with response within tumour compartments. Univariate overall-survival (OS) analysis of stromal proteins supported these findings, with ER-alpha expression (HR=0.19, p=0.0012) associated with better OS while MART1 expression (HR=2.3, p=0.035) was indicative of poorer OS. Proteins within tumour compartments consistent with longer OS included PD-L1 (HR=0.53, p=0.023), FOXP3 (HR=0.5, p=0.026), GITR (HR=0.51, p=0.036), SMA (HR=0.59, p=0.043), while EPCAM (HR=1.7, p=0.045), and CD95 (HR=4.9, p=0.046) expression were associated with shorter OS. Our data provides early insights into the levels of these markers in the TNBC tumour microenvironment, and their association with chemotherapeutic response and patient survival., Competing Interests: NM is employed by QCIF Bioinformatics. The remaining 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 Kulasinghe, Monkman, Shah, Matigian, Adams and O’Byrne.)

Breast cancer is a leading cause of cancer-associated death in women. The clinical management of breast cancers is normally carried out using a combination of chemotherapy, surgery and radiation therapy. The majority of research investigating breast cancer therapy until now has mainly utilized two-dimensional (2D) in vitro cultures or murine models of disease. However, there has been significant uptake of three-dimensional (3D) in vitro models by cancer researchers over the past decade, highlighting a complimentary model for studies of radiotherapy, especially in conjunction with chemotherapy. In this review, we underline the effects of radiation therapy on normal and malignant breast cells and tissues, and explore the emerging opportunities that pre-clinical 3D models offer in improving our understanding of this treatment modality.

Barrier-to-Autointegration Factor 1 (Banf1/BAF) is a critical component of the nuclear envelope and is involved in the maintenance of chromatin structure and genome stability. Banf1 is a small DNA binding protein that is conserved amongst multicellular eukaryotes. Banf1 functions as a dimer, and binds non-specifically to the phosphate backbone of DNA, compacting the DNA in a looping process. The loss of Banf1 results in loss of nuclear envelope integrity and aberrant chromatin organisation. Significantly, mutations in Banf1 are associated with the severe premature ageing syndrome, Néstor-Guillermo Progeria Syndrome. Previously, rare human variants of Banf1 have been identified, however the impact of these variants on Banf1 function has not been explored. Here, using in silico modelling, biophysical and cell-based approaches, we investigate the effect of rare human variants on Banf1 structure and function. We show that these variants do not significantly alter the secondary structure of Banf1, but several single amino acid variants in the N- and C-terminus of Banf1 impact upon the DNA binding ability of Banf1, without altering Banf1 localisation or nuclear integrity. The functional characterisation of these variants provides further insight into Banf1 structure and function and may aid future studies examining the potential impact of Banf1 function on nuclear structure and human health., Competing Interests: The authors declare competing financial interests; EB, DR, and KO. are founders of Carpe Vitae Pharmaceuticals. EB, KO, DR, JB, and MA. are inventors on provisional patent applications filed by Queensland University of Technology. The remaining 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 © 2021 Rose, Bai, Tang, Cheong, Beard, Burgess, Adams, O’Byrne, Richard, Gandhi and Bolderson.)