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A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. A variety of fluorescence signal removal methods were also evaluated, where incorporation of a photocleavable link (PCL) resulted in full fluorescence signal removal with minimal tissue disruption. In summary, this work resulted in an optimized Ab-oligo cyCIF platform capable of generating high dimensional images to characterize the spatial proteomics of the hallmarks of cancer.

Background In Australian Aboriginal and Torres Strait Islander communities, childcare is traditionally shared by kin. Little is known about how grandparental care impacts Aboriginal child health and evidence from other countries is mixed. We explored relationships between grandparental health (a proxy for grandparental care) and health and health service use by Aboriginal children born in Western Australia from 2000 to 2013. Methods This is a retrospective cohort study using linked administrative health data. Outcomes were child mortality, hospital admissions, and emergency department (ED) presentations up to five years old. Grandparental health when the child was born was categorised as healthy (none/one Elixhauser condition), unhealthy (two or more conditions), or deceased. Grandparental-child health associations were estimated using regression with adjustment for birth year, sex, remoteness, socioeconomic advantage, maternal smoking, and maternal age. Results 29,409 Aboriginal children linked to their maternal grandmothers. 70% also linked to maternal grandfathers, 66% to paternal grandmothers, and 49% to paternal grandfathers. 86% of maternal grandmothers were healthy, 7% unhealthy, and 7% deceased. Children with healthy grandmothers had an average of 27% fewer hospital days (adjusted incidence rate ratio: 0.73, 95% CI: 0.65, 0.83) than those with deceased grandmothers. They also had lower mortality and fewer potentially avoidable admissions and emergency department presentations. Children with unhealthy or deceased grandmothers had comparable rates. These patterns were similar for all four grandparents, but generally stronger for maternal grandmothers. Stillbirth and unavoidable admissions were unrelated to grandparental health. Conclusions Aboriginal children with healthy grandparents had better health and lower health service use. If this relationship is causal, healthy ageing and strong family connections must be supported to improve child health. Even if the relationship is not causal, healthy ageing, a family-centred approach to health care, and social support may help families experiencing poor health in multiple generations simultaneously.

Persistently high, worldwide mortality from cancer highlights the unresolved challenges of disease surveillance and detection that impact survival. Development of a non-invasive, blood-based biomarker would transform survival from cancer. We demonstrate the functionality of ultra-high content analyses of a newly identified population of tumor cells that are hybrids between neoplastic and immune cells in patient matched tumor and peripheral blood specimens. Using oligonucleotide conjugated antibodies (Ab-oligo) permitting cyclic immunofluorescence (cyCIF), we present analyses of phenotypes among tumor and peripheral blood hybrid cells. Interestingly, the majority of circulating hybrid cell (CHC) subpopulations were not identified in tumor-associated hybrids. These results highlight the efficacy of ultra-high content phenotypic analyses using Ab-oligo based cyCIF applied to both tumor and peripheral blood specimens. The combination of a multiplex phenotypic profiling platform that is gentle enough to analyze blood to detect and evaluate disseminated tumor cells represents a novel approach to exploring novel tumor biology and potential utility for developing the population as a blood-based biomarker in cancer.

Significance: Advanced genetic characterization has informed cancer heterogeneity and the challenge it poses to effective therapy; however, current methods lack spatial context, which is vital to successful cancer therapy. Conventional immunolabeling, commonplace in the clinic, can provide spatial context to protein expression. However, these techniques are spectrally limited, resulting in inadequate capacity to resolve the heterogenous cell subpopulations within a tumor. Aim: We developed and optimized oligonucleotide conjugated antibodies (Ab-oligo) to facilitate cyclic immunofluorescence (cyCIF), resulting in high-dimensional immunostaining. Approach: We employed a site-specific conjugation strategy to label antibodies with unique oligonucleotide sequences, which were hybridized in situ with their complementary oligonucleotide sequence tagged with a conventional fluorophore. Antibody concentration, imaging strand concentration, and configuration as well as signal removal strategies were optimized to generate maximal staining intensity using our Ab-oligo cyCIF strategy. Results: We successfully generated 14 Ab-oligo conjugates and validated their antigen specificity, which was maintained in single color staining studies. With the validated antibodies, we generated up to 14-color imaging data sets of human breast cancer tissues. Conclusions: Herein, we demonstrated the utility of Ab-oligo cyCIF as a platform for highly multiplexed imaging, its utility to measure tumor heterogeneity, and its potential for future use in clinical histopathology.

Positive margin status due to incomplete removal of tumor tissue during radical prostatectomy for high-risk localized prostate cancer requires reoperation or adjuvant therapy, which increases morbidity and mortality. Adverse effects of prostate cancer treatments commonly include erectile dysfunction, urinary incontinence, and bowel dysfunction, making successful initial curative prostatectomy imperative. Current intraoperative tumor margin assessment is largely limited to frozen section analysis, which is a lengthy, labor-intensive process that is obtrusive to the clinical workflow within the operating room (OR). Therefore, a rapid method for prostate cancer margin assessment in the OR could improve outcomes for patients. Dual probe difference specimen imaging (DDSI), which uses paired antibody-based probes that are labeled with spectrally distinct fluorophores, was shown herein for prostate cancer margin assessment. The paired antibody-based probes consisted of a targeted probe to prostate-specific membrane antigen (PSMA) and an untargeted probe, which were used as a cocktail to stain resected murine tissue specimens including prostate tumor, adipose, muscle, and normal prostate. Ratiometric images (i.e., DDSI) of the difference between targeted and untargeted probe uptake were calculated and evaluated for accuracy using receiver operator characteristic curve analysis with area under the curve values used to evaluate the utility of the DDSI method to detect PSMA positive prostate cancer. Targeted and untargeted probe uptake was similar between the high and low PSMA expressing tumor due to nonspecific probe uptake after topical administration. The ratiometric DDSI approach showed substantial contrast difference between the PSMA positive tumors and their respective normal tissues (prostate, adipose, muscle). Furthermore, DDSI showed substantial contrast difference between the high PSMA expressing tumors and the minimally PSMA expressing tumors due to the ratiometric correction for the nonspecific uptake patterns in resected tissues. Previous work has shown that ratiometic imaging has strong predictive value for breast cancer margin status using topical administration. Translation of the ratiometric DDSI methodology herein from breast to prostate cancers demonstrates it as a robust, ratiometric technique that provides a molecularly specific imaging modality for intraoperative margin detection. Using the validated DDSI protocol on resected prostate cancers permitted rapid and accurate assessment of PSMA status as a surrogate for prostate cancer margin status. Future studies will further evaluate the utility of this technology to quantitatively characterize prostate margin status using PSMA as a biomarker.

Background Evidence suggests that Aboriginal babies in Western Australia are not receiving adequate primary health care in their first 3 months of life, leading to questions about enablers and constraints to delivering such care. This paper presents findings from a qualitative research project investigating health providers’ perceptions and experiences of best and current practice in discharge planning, postnatal care and health education for Aboriginal mothers and their newborn babies. Methods Constructivist grounded theory guided this research involving 58 semi-structured interviews conducted with health providers who deliver care to Aboriginal mothers and infants. Participants were recruited from hospital-based and primary health sites in metropolitan Perth, and regional and remote locations in Western Australia. Results Structural factors enabling best practice in discharge planning, postnatal care, and health education for mothers included health providers following best practice guidelines and adequate staffing levels. Organisational enablers included continuity of care throughout pregnancy, birth and postnatally. In particular, good communication between services around discharge planning, birth notifications, and training in culturally respectful care. Structural and organisational constraints to delivering best practice and compromising continuity of care were identified as beyond individual control. These included poor communication between different health and social services, insufficient hospital staffing levels leading to early discharge, inadequate cultural training, delayed receipt of birth notifications and discharge summaries received by Aboriginal primary health services. Conclusion Findings highlight the importance of examining current policies and practices to promote best practice in postnatal care to improve health outcomes for mothers and their Aboriginal babies.

Background Aboriginal infants have poorer birth outcomes than non-Aboriginal infants. Harmful use of tobacco, alcohol, and other substances is higher among Aboriginal women, as is violence, due to factors such as intergenerational trauma and poverty. We estimated the proportion of small for gestational age (SGA) births, preterm births, and perinatal deaths that could be attributed to these risks. Methods Birth, hospital, mental health, and death records for Aboriginal singleton infants born in Western Australia from 1998 to 2010 and their parents were linked. Using logistic regression with a generalized estimating equation approach, associations with birth outcomes and population attributable fractions were estimated after adjusting for demographic factors and maternal health during pregnancy. Results Of 28,119 births, 16% of infants were SGA, 13% were preterm, and 2% died perinatally. 51% of infants were exposed in utero to at least one of the risk factors and the fractions attributable to them were 37% (SGA), 16% (preterm) and 20% (perinatal death). Conclusions A large proportion of adverse outcomes were attributable to the modifiable risk factors of substance use and assault. Significant improvements in Aboriginal perinatal health are likely to follow reductions in these risk factors. These results highlight the importance of identifying and implementing risk reduction measures which are effective in, and supported by, Aboriginal women, families, and communities.

Background The prevalence of mental illness, substance use disorders, and their dual diagnosis is disproportionately high among people in prisons compared to the community. Accurate prevalence estimates are required to inform resourcing of prison health services and reduce the risk of harm to people experiencing these conditions. Existing estimates, where available, often rely on only one data source. Method We used three data sources – self-reported history of diagnoses, in-prison medical records, and administrative data to estimate the prevalence of mental illness, substance use disorder, and dual diagnosis among two large cohorts of non-Indigenous and Aboriginal and Torres Strait Islander people in Australian prisons. We calculated population-weighted proportions of the samples with each condition. Inter-rater reliability metrics inform data source agreement. Results The prevalence of mental illness only, substance use disorder only, and dual diagnosis was 17.0% (95%CI 12.0–24.5), 14.8% (95%CI 9.6–18.1), and 44.2% (95%CI 33.2–54.7), respectively, for incarcerated, non-Indigenous adults. For incarcerated Aboriginal and Torres Strait Islander adults, our corresponding estimates were 7.0% (95%CI 4.3–11.5), 26.8% (95%CI 18.9–33.5), and 40.9% (95%CI 30.1–48.2). These estimates differed significantly from those derived from singular data sources. Individual data sources’ agreement was weakest for substance use disorder diagnoses and strongest for dual diagnoses. Conclusions Individual data sources likely have high specificity and low sensitivity, thus under-ascertaining diagnoses. We recommend using multiple data sources to estimate prevalence to ensure adequate ascertainment of these conditions among people in prison and to ensure in-prison and transitional health services are appropriately resourced.

Advances in our understanding of the complex, multifaceted interactions between tumor epithelia, immune infiltrate, and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss—particularly in fragile samples—, and challenges with antibody labeling. Herein, we developed and optimized an oligonucleotide antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity. We also extended the oligonucleotide barcoding strategy to secondary antibodies to enable the inclusion of difficult-to-label primary antibodies in a cyCIF panel. Using both the amplification oligonucleotides to label DNA barcoded antibodies and in situ hybridization of multiple fluorescently labeled oligonucleotides resulted in signal amplification and increased signal-to-background ratios. This procedure was optimized through the examination of staining parameters including staining oligonucleotide concentration, staining temperature, and oligonucleotide sequence design, resulting in a robust amplification technique. As a proof-of-concept, we demonstrate the flexibility of our cyCIF strategy by simultaneously imaging with the original oligonucleotide conjugated antibody (Ab-oligo) cyCIF strategy, the novel Ab-oligo cyCIF amplification strategy, as well as direct and indirect immunofluorescence to generate highly multiplexed images.