Abstract
Bladder cancer (BC) is the 10th most common cancer world-wide with an estimated 570,000+ people
being diagnosed in 2020. BC can be broadly subdivided into non-muscle invasive (NMI) and muscle
invasive (MI) BC, with further subdivisions of low-grade and high-grade disease. A common
immunotherapeutic treatment for HG NMIBC is intravesical Bacillus Calmette-Guerin (BCG) vaccine,
following tumour resection. Although this post-surgical instillation regime has shown to dramatically
reduce the likelihood of disease recurrence, some 30-40% of patients fail to clinically respond to BCG
therapy. Additionally, the exact immunotherapeutic mechanism and factors affecting efficacy are yet
to be fully elucidated. From this, we postulate that gaining a deeper understanding of the tumour
microenvironment, via a holistic multi-omics approach, will provide valuable insight into these factors.
Bacterial signatures within patient catheterised urine (n=67) and FFPE tissues (n=72), (matching
patients n=44), were determined using 16s rRNA sequencing, processed through QIIME2 and clustered
into Operational Taxonomic Units (OTUs). Alpha (Shannon and observed) and Beta (Bray-Curtis)
diversity analysis was performed. RNA extracted from FFPE BC tissues (n=106) generated gene
expression data using the Nanostring IO360 panel (770 gene CodeSet). 9-colour multiplex
immunohistochemistry (mIHC) using PhenoimagerHT (Akoya Biosciences) was performed to
investigate and spatially define immune cell types within the TME (n=75). Urine specimens (n=69)
underwent investigation of 23 cytokines using the IsoSpark proteomics systems (IsoPlexis). Patient
FFPE tissues and urines were derived from Royal Surrey County Hospital, Guildford, UK, with medical
histories, questionnaires, and NHS histopathologist assistances for sample validation.
Results revealed via using mIHC, that BCG non-responders may have an innate immunosuppressive
TME (increased PD-L1+, higher Tregs counts, closer proximities of Treg:CD8+ and Treg:NK cells) at
baseline before BCG immunotherapy compared to BCG responders. Gene expression data suggests
that genes involved in mast cell activation (SPP1 and C5AR1) may have a role in BCG response, with
differential gene expression of samples positive for LPS (bacterial marker) showing an upregulation in
mast cells count. Bacterial analysis showed a decrease in bacterial diversity with increasing disease
progression, alongside an increased population of anaerobic taxa. Urine specimens was shown to be
an unreliable proxy for the bacteria composition in matched FFPE tissues. Linear discrimination
analysis showed an increase in Pseudomonas, Vibrio and Kocuria, amongst others in the treatment
naïve known BCG responder cohort. Cytokine analysis suggests BCG responders have elevated levels
of various pro-inflammatory cytokines at baseline compared to BCG non-responders.
Ultimately, these findings will aid in our understanding of the BC TME and possibly lead to better
patient treatment stratification, novel therapeutic targets, and improved therapeutic outputs.