BOB Ped Surg 2023 - Bernadette Jeremiasse, IPSO - Presentation

Achieving complete tumor resections without major complications remains challenging because some tumors are difficult to distinguish from healthy tissue and in other tumors the margin is very narrow.

Fluorescence guided surgery with tumor-specific probes can visually assist the surgeon in discriminating tumors from healthy tissue.

Tumor-specific probes are currently screened one by one and per tumor type.

The imaging platform combines tumor organoid biobanks with 3D live imaging and is able to screen fluorescent guided surgery probes in a tumor-specific and patient-specific way.

Organoids maintain molecular tumor characteristics and can be held in culture for a long time.

The platform uses both adult and pediatric tumor organoid biobanks, specifically neuroblastoma and breast cancer.

Control organoids were chosen based on the tissue surrounding the tumor: kidney organoids for neuroblastoma and healthy breast organoids for breast cancer.

Surface markers upregulated in neuroblastoma or breast cancer were identified based on RNA profiling, the human protein atlas, and literature review.

Probes targeting surface markers were conjugated to six different fluorophores ranging between 488 and 647 nanometers.

Seven-colored 3D live imaging using confocal microscopy on living organoids was performed, including six probe fluorophores plus the general marker eFluor.

Segmentation analysis was performed using the STAPLE pipeline developed in the laboratory, and fluorescent signals were quantified.

The three most promising probes for neuroblastoma were validated in vivo using a mouse xenograft model with tumors originating from neuroblastoma organoids.

Probe fluorescence varies between patients and even within a patient in neuroblastoma organoid lines.

Different probes work on tumor organoids compared to healthy control organoids.

The STAPLE pipeline performs 3D segmentation by first segmenting membrane and nuclear signals, generating cells, and combining cells to form single organoids.

An organoid is determined to be positive when the intensity of the tumor organoid is at least twice the fluorescence of the healthy control organoid.

The order of neuroblastoma organoid line performance was similar in vitro and in vivo, with line 129 being highest and line 67 being lowest.

One of the tested probes is advancing to a clinical trial this year.

Thousands of organoids were quantified using this methodology.

Four markers showed promise for neuroblastoma based on high percentages of positive organoids and high fluorescent intensities.

The platform can predict which combinations of probes are most promising by screening six probes simultaneously.

The three most promising probes tested in vivo for neuroblastoma were GD2, L1CAM, and NCAM.

These three probes resulted in tumor-to-background ratios above 2, indicating good intraoperative visibility.

In vitro imaging patterns resemble the efficacy in vivo, with L1CAM showing higher signal than GD2 in both settings.