In the following work, we describe the consecutive steps necessary for the establishment of a large biobank of colorectal and pancreatic cancer.
In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested.
Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the “donor” tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.
In recent years, the accumulated knowledge of cancers' morphologic, clinical and genetic properties led to the conception of cancer as a heterogeneous, individual disease. Consequently, mutational characterization of neoplasms, besides clinical and pathological features, has gained importance for clinical decision making and many targeted therapies were developed for various molecular alterations. For instance, the efficacy of cetuximab in colorectal cancer treatment can be predicted by the analysis of the KRAS and PIK3CA mutational status1. Precision medicine aims for a tailored approach to provide the highest treatment response in each patient and avoid toxicity of inefficacious therapies2. Biobanks contain tissue, blood and other biological materials of cancer patients, which are linked to the clinical data, and thus are an excellent tool for translational cancer research. Due to the large number of clinical samples, biobanks enable the detection of rare, but potentially druggable mutations, which provides new treatment opportunities for the individual patient3.
To cover as broad as possible an oncologic research spectrum, we did not restrain our activity on sample harvesting alone, but focused on the establishment of patient-derived cancer cell lines and xenografts (PDX). Traditional 2D cell lines remain the corner stone of in vitro research and are the prime choice for large scale drug screenings4,5. Moreover, cell line analysis is often easier, cheaper and more readily available. Additionally, since patient-derived peripheral blood lymphocytes (PBL) are available, also tumor immunology can be studied in vitro6. However, the majority of newly developed drugs with promising preclinical effectivity in cell based in vitro or in vivo experiments, have shown disappointing results in clinical trials7. In contrast, preclinical studies based on PDX in vivo studies have reflected the clinical activity of antineoplastic agents much more faithfully8. Since PDX tissue closely reflects the histological and molecular properties of the donor tumor, PDX models are a good way to propagate the often very limited amounts of viable tumor tissue to maintain the integrity of a biobank and to allow the exchange of samples between research groups and institutions. Moreover, cancer cell lines derived from PDX tissue can be established significantly easier than primary cancer cell lines9. In recent years, our working group has established a comprehensive integrated colorectal and pancreatic cancer biobank by stepwise standardizing and optimizing the work flow for all biological samples in question (Figure 1).
Figure 1: Workflow and organization of the biobank Please click here to view a larger version of this figure.
The following study has been approved by the institutional review board of the University Medical Center Rostock (II HV 43/2004, A 45/2007, A 2018-0054, A 2019-0187 and A 2019-0222). Furthermore, all veterinary relevant procedures have been approved by the Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern under the registration numbers LALLF M-V/TSD/ 7221.3-2-020/17 and 7221.3-1-007/19.
1. Experimental Prerequisites
2. Sample collection
3. Serum processing
4. Isolation of PBL by density gradient centrifugation
NOTE: Work parallel with each of the two 20 mL syringes.
5. Tissue processing
NOTE: Start with the generation of snap frozen samples of tumor and healthy tissue to maintain the integrity of nucleic acids.
6. Primary cell culture
7. PDX Generation
8. PDX harvesting and processing
9. Biobank and data management
Laboratory location/name | cancer entity | consecutive case number | specification | consecutive number |
C=colorectal | _Met=Metastasis | |||
P=pancreatic | _Tu=Tumor | |||
Example: HROC389_Met2 = Rostock, colorectal cancer, case 389, second metastasis |
Table 1: Definition of the sample ID.
Tumor-ID | Prior storage in N2 (=f) | Passage (=T) number | consecutive mouse (=M) number |
Example: HROP12 fT0 M1 = Rostock, pancreatic cancer, case 12, generated from frozen primary tissue, first passage, mouse 1. |
Table 2: Definition of the PDX ID.
In our hands, the establishment rate of primary cell cultures (Figure 2A & B) was 12.9% in a large series9. The majority of attempts to isolate expandable tumor cells from fresh surgical resected specimens failed due to a lack of outgrowth or early contamination. Cell line establishment was considered successful after 3 passages with a steady growth under standard culture conditions (DMEM, 10% FCS, standard culture vessel) and validation of epithelial differentiation via FACS-analysis10. Cell lines derived from PDX tumors (Figure 2C & D) showed a higher establishment rate of 23.6% which is also due to the possibility of repetitive attempts in contrast to primary resected tumors9. However, some mixed cultures (Figure 2E) cannot be freed of fibroblastic growth or are even lost due to fibroblastic overgrowth (Figure 2F).
Figure 2: Cell culture. Primary cancer cell lines, derived from a metastasis of colon cancer case HROC313, passage 21 (A) and pancreatic cancer case HROP88, passage 5 (B). PDX-derived cancer cell lines of colon PDX HROC285 T0 M2 (D) and pancreatic PDX HROP10 T5 M2, passage 4 (E). Mixed culture of fibroblasts and cancer cells from pancreatic cancer HROP75, passage 8 (C) and fibroblastic overgrowth (F). Please click here to view a larger version of this figure.
Considering changes in PDX generation protocol, mouse strains used and also experimenters over several years, as well as large differences in the amount of tumor tissue available for engraftment, it is not trivial to give the overall success rate of PDX generation. In a very recent series of PDX generation experiments performed by two researchers (S.M. and F.B.), primary outgrowth rates of 63% for colorectal PDX (an exemplary histology can be depicted from Figure 3A) and 48% for pancreatic PDX (Figure 3B) were observed. The outgrowth of murine or human lymphomas at the implantation site is relatively rare, but can mimic successful PDX outgrowth (Figure 3C). Apart from histopathological examination, concordance between PDX models and their donor patients was regularly confirmed by short tandem repeat (STR) analysis (Figure 3D). To the present day the biobank comprises >50 primary and >50 secondary colorectal, 3 primary and 6 secondary pancreatic cancer cell lines as well as >150 colorectal and 19 pancreatic PDX models.
Figure 3: Representative histological comparison of colorectal (A) and pancreatic PDX (B). Human lymphoma at the implantation site mimicking PDX outgrowth (C). Genetic identity testing of a PDX model (HROC430 T1 M2) to the original patient tumor tissue (HROC430Tu) by short tandem repeat (STR) analysis. Comparison of the nine STR loci, vWA, THO1, TPOX, CSF1 PO (FAM dye) and D5S818, D13S317, D7S820, D16S539 (HEX dye) using multiplex PCR with fluorescent-labeled primers following capillary electrophoresis confirmed genetic concordance of the PDX and donor tumor (D). Please click here to view a larger version of this figure.
The generation of a living biobank presupposes, apart from complying with the legal regulations of privacy, medical law and animal welfare, a good infrastructure and a well-coordinated team. It has proven advantageous to directly involve a part of the surgical staff in the research procedures, since they can very well assess the suitability of the individual patient for tissue donation. Moreover, patients tend to consent with biobanking more frequently, when their written approval is obtained within the course of the surgical informed consent discussion. To save time and resources, cases that will presumably yield insufficient amounts of tumor tissue should not be selected for biobanking. When it comes to specimen acquisition, the maxim "communication is key" is a simple, but often overlooked truth. It only takes a single uninformed theatre nurse or surgical colleague to ruin the specimen right at the outset by proceeding as usual and adding formaldehyde to the resection specimen. Therefore, it is absolutely crucial that every single member of the involved staff gets acquainted with the SOP for biobanking. Surgeons should be noticed the day before and right at the start of the procedure about scheduled tissue collection. Furthermore, cases selected for biobanking, should be highlighted in the electronic OR plan. Tissue harvesting from the surgical specimen should be performed by a pathologist. First, this will ensure that the tissue harvesting does not interfere with the final pathological report. Second, this increases the probability of receiving tissue with adequate amounts of viable cancer tissue. Especially in pancreatic cancers with a pronounced desmoplastic reaction and frequent necrotic areas, viable parts are hard to identify macroscopically for the untrained eye. As an exception to this rule, tissue blocks from large hepatic or pulmonal metastases, may at times be excised "back-table" by the surgeon, if surgical margins can be defined macroscopically. Rectal cancer resected by total mesorectal excision (TME), might not be suitable for biobanking, since tissue harvesting from the resected specimen prior to paraffin embedding might interfere with the TME quality assessment. Alternatively, tissue for biobanking can be acquired by transanal biopsy of rectal cancer.
The establishment rates for primary cell cultures derived from the original tumor are generally low. PDX-derived, secondary cell cultures can more likely be successfully established.We recommend testing of different media for each case and use of antibiotic supplements for the first passages to reduce contamination to a minimum since the harvested tissue is rarely sterile. After successful propagation, each individual cell line should be confirmed as a cancer cell line by FACS analysis and regularly tested for mycoplasma contamination. To exclude cross-contamination, regular STR analysis is advisable. It should be noted, that the establishment protocol for primary and secondary cell lines is constantly subjected to optimization. Details concerning the composition and success rates of the single media are clearly beyond the scope of this work and will be published separately.
For PDX engraftment, tumor tissue can be either implanted directly after resection or cryopreserved in fetal calve serum with 10% DMSO or similar freezing media for delayed implantation. Implantation immediately upon tumor tissue harvesting puts a strain on logistics and laboratory staff, and xenografting results after cryopreservation are not inferior at all 10. Moreover, incubation of the tissue in Matrigel prior to tumor implantation, significantly increases engraftment rates12. We recommend delayed engraftment following definite pathological finding and immediate disposal of erroneously collected tissue specimens. Since the success rate of primary engraftment increases with immunodeficiency of the recipient mouse, we tend to use NSG mice for the very first PDX passage. After the first successful PDX engraftment, NMRInu/nu mice can and should be used for subsequent passages and tissue expansion. This strain is more robust, cheaper and easier to breed compared to NSG or similar immunodeficient strains, but still shows reasonable engraftment rates. Moreover, its nudeness facilitates implantation and tumor growth monitoring. To increase the engraftment rates in subsequent passages, we recommend direct transfer of freshly harvested PDX tissues to host mice whenever possible, especially for slow growing PDX and cases with a low primary engraftment success rate. Collins and Lang recently reviewed 14 studies of colorectal PDX establishment and reported engraftment rates varying from 14 to 100% with a median PDX establishment rate of 68%, the latter being consistent with our findings13. In line with the literature, we observed lower establishment rates of pancreatic compared to colorectal cancer PDX14. Regardless of the host mouse strain and tumor entity, the outgrowth of human, Epstein-Barr Virus (EBV)-associated B-cell lymphomas and murine lymphomas at the implantation side poses an important pitfall15,16. If unrecognized, such tumors can "contaminate" subsequent passages and thus confound consecutive results. Unusual fast PDX growth and swelling of cervical, axillar and inguinal lymph nodes are strong indicators of murine lymphoma growth, but regular histological examination of PDX is nevertheless advisable. Furthermore, genetic concordance between PDX and the corresponding donor patient should be tested regularly by STR-analysis. Ideally, the biobank should be linked to a clinical database comprising patients characteristics (general information, survival, relapse free survival, therapy, secondary neoplasia etc.). Due to legal regulations of privacy protection and lack of such an anonymized data base, our clinical data set is regularly administrated and updated manually by the cooperating physicians.
While conventional biobanks are limited to observatory research, a living biobank provides the opportunity for in vitro and in vivo interventions. Patient-derived cell lines are an important tool for fundamental research, high-throughput drug screenings and assessment of new pharmaceutical agents4. Corresponding PDX models, however, are of increasing importance, since they closely recapitulate the histology of the original tumor17,18 and show a high genetic stability over several passages19,20. Our PDX biobank has proven itself as an excellent platform for preclinical and fundamental research6,21. Moreover, since large PDX collections adequately reflect the inter-individual heterogeneity of the patient population, the PDX clinical trial (PCT) approach (one animal per model per treatment) has gained importance for drug development since it allows the faithful prediction of clinical response to new drugs and combinatorial regimen8. We also are currently evaluating new experimental drugs in small PCT trials.
Despite these promising results, the median establishment duration of 12.2 month, impedes the clinical applicability of PDX models as "avatar mice" for testing anticancer treatment options, at least for those patients in need of immediate adjuvant or even neoadjuvant treatment22. An additional disadvantage of standard PDX models is the lack of usability for immunotherapy testing due to host mice's immunodeficiency. To overcome these limitations, several "humanized" mouse strains have been developed. These mice are heavily immunocompromised, but can be reconstituted with various types of human bone marrow-derived cells or CD34+ hematopoietic stem cells subsequent to PDX outgrowth23, allowing the evaluation of lymphocyte-mediated cytotoxicity and of therapy response to immune checkpoint inhibitor treatment24,25.
In recent years, patient-derived organoids (PDO) emerged as important cancer models competing with PDX. Derived from intact tumor pieces and cultured in an extracellular matrix scaffold, these three-dimensional structures closely reflect the histologic and genetic properties of the original tumor. The possibility of long-term expansion and cryopreservation renders PDO an ideal supplement of a living biobank26,27. In addition to a relatively high establishment rate, reliable drug response prediction has been reported for PDO of several tumor entities28. Moreover, PDOs have even been generated from circulating tumor cells and also the simultaneous establishment of organoids from corresponding healthy tissue is possible, allowing assessment of therapy-related toxicity on a patient-individual basis29,30. However, compared to conventional 2D cell cultures, organoid culture is time and resource consuming and artificial extracellular matrix compounds can interfere with certain analytic procedures31. Moreover, cancer organoids are susceptible to overgrowth by faster growing, non-malignant organoids derived from healthy epithelium30. Due to a lack of stroma, blood vessels and immune cells, PDOs are mostly inapplicable for the testing of antiangiogenic immunotherapeutic agents. Yet, new culturing methods allow the modeling of tumor microenvironment in vitro, rendering PDOs a true contender for PDX models32. In the near future, patient-individual tumor models, combined with powerful genetic tools like next-generation sequencing, will hopefully pave the path to true precision medicine and tailored-treatment approaches.
The authors have nothing to disclose.
We kindly acknowledge Jenny Burmeister, our graphical assistant, for the recording and editing of the video. Furthermore, we thank our colleagues of the surgical and pathological department for the longstanding collaboration. We would also like to thank Marcus Müller, production manager of the IT and Media Centre, University of Rostock, for supplying the audio recording equipment and refining the sound quality.
FUNDING: The German Cancer Aid Foundation (DKH e.V.), grant number 108446, and grant number TBI-V-1-241-VBW-084 from the state Mecklenburg-Vorpommern partly funded this research.
Bacillol® AF; 1L | Bode, Hartmann | REF 973380 | desinfection |
PP centrifuge tube, 15ml; sterile | Greiner Bio One | GBO Cat. No.:188271 | centrifuge tube |
PP centrifuge tube, 50ml, sterile | Sarstedt | Order number: 62.547.254 | centrifuge tube |
BD DiscarditTM II Syringe 20ml | BD | REF 300296 | blood collection |
Serum 7,5ml Sarstedt Monovette | Sarstedt | Item number: 01.1601 | blood collection |
serological Pipette 10ml | Sarstedt | REF 86.1254.001 | liquid transfer |
Pipetboy ratiolab® accupetta | Ratiolab | Item number: RL3200300 | liquid transfer |
PIPETBOY acu 2 | Integra Biosciences | VWR Cat.No: 613-4438 | liquid transfer |
DPBS; w/o Ca & Mg | Pan Biotech | Cat. No.: P04-36500 | washing |
Pancoll human | Pan Biotech | Cat. No.: P04-60500 | density gradient centrifugation |
DMEM/F12 (Dulbecco’s Modified Eagle Medium) | PAN Biotech | Cat. No.: P04-41500 | cell cultivation |
FBS Good Forte (Filtrated Bovine Serum) | PAN Biotech | Cat. No.: P40-47500 | cell cultivation |
L-Glutamine 200mM | PAN Biotech | Cat. No.: P04-80100 | cell cultivation |
Trypsin / EDTA | PAN Biotech | Cat. No.: P10-023100 | cell cultivation |
DMSO (Dimethyl Sulfoxid for cell culture) | PanReac AppliChem | VWR Cat.No: A3672.0250 | cell freezing |
Freezer Medium (FCS with 10% DMSO) | selfmade | — | cell freezing |
cryotube- CryoPure 2ml | Sarstedt | 72380 | cell freezing |
6-Well cell culture plate; steril; with lid | Greiner bio-one | Cat.-No.: 657 160 | cell cultivation |
Petri dish 92 x 16 mm, PS, without cams | Sarstedt | Cat. No.: 82.1472.001 | tissue preparation |
sterile surgical blades | B.Braun (Aesculap) | REF BB510 | tissue preparation |
BD DiscarditTM II Syringe 10ml | BD | REF 309110 | tissue preparation |
cell strainer; yellow; 100µm | Falcon | REF 352360 | tissue preparation |
CoolCell | biocision | Item number: 210004 | cooling container with -1°C/min |
Dewar transport vessel type 27 B, 2 l, 138 mm | KGW | Cat. No.: HT39.1 | transport system |
Pipette tip 200µl | Sarstedt | REF 70.760.002 | liquid transfer |
Filter tip 1000µl | Sarstedt | REF 70.762.411 | liquid transfer |
Pipette 200µl, yellow | Eppendorf | Cat. No.: 3121 000.082 | liquid transfer |
Pipette 1000µl, blue | Eppendorf | Cat. No.: 3121 000.120 | liquid transfer |
incubator BB 6220 CU | Heraeus | Cat.-No.: 51012839 | cell cultivation |
heating plate PRÄZITHERM | Harry Gestigkeit GmbH | — | heating |
Microscope Zeiss Primo Vert | Carl Zeiss MicroImaging GmbH | Serial number. 3842000839 | imaging cell cultures |
Sterile bench Safe flow 1.8 nunc | nunc GmbH & Co. KG | — | sterile working bench |
freezer -80°C | Kryotec-Kryosafe GmbH | — | sample storage |
Electronic balance MP-300 | Chyo | — | Scale |
BD Micro-fine, U100 insulin syringe | BD | REF 324826 | injection anesthetic |
Rompun 2%; 25ml | Bayer | approval number: 6293841.00.00 | anesthesia |
Ketamin 100 mg/ml, 25ml | CP-Pharma GmbH | approval number: 401650.00.00 | anesthesia |
GES3S Reader | Datamars | not available | RFID reader |
ISO-Transponder FDX-B (1,4x8mm) | Peddymark | — | RFID chip |
Cotrim-ratiopharm® Ampullen SF 480 mg/5 ml | Ratiopharm | PZN-03928197 | antibiotic drinking water |
Heating plate #FM-20 42x28cm | Dragon | — | heating |
Heating lamp | Electric Petra, Burgau | — | heating |
Ointment for the eyes and nose (5% Dexpanthenol) Bepanthen | Bayer | PZN-01578675 | Eye protection |
anatomical tweezer | B.Braun Aesculap | BD21 OR | surgical instruments |
surgical tweezer | B.Braun Aesculap | BD50 1 R | surgical instruments |
scissors | B.Braun Aesculap | BC05 6R | surgical instruments |
needle holder | B.Braun Aesculap | BH1 1 OR | surgical instruments |
Prolene 5-0 | Ethicon | XN8870.P32 | surgical suture material |
Opsite moisture vapour permable spray dressing | Smith&Nephew | REF 66004978, PZN- 02063507 | surgical suture material |
Adhesive aperture drape | Barrier | REF 904622 | sterile OP tissue |
gauze swap Gazin®; steril; 10×10 cm | Lohmann&Rauscher | REF 18506 | sterile OP tissue |
Raucotupf cotton tipped applicators | Lohmann&Rauscher | REF 11969 | applicator |
Corning® Matrigel Basement Membrane Matrix | Corning | Cat.-No.: 354234 | Basement Membrane Matrix |
iodine solution Braunol (7,5g povidone iodine) | B.Braun Melsungen AG | Item number: 18839 | desinfection |
MACS® Tissue Storage Solution | Miltenyi Biotec GmbH | Order No.:130-100-008 | storage solution |
Formafix 4% | Grimm med. Logistik GmbH | Item number: F10010G | fixation solution |
Software FreezerworksBasic | Dataworks Development, Inc | — | sample organization |
Zebra TLP 2844 printer | Zebra | — | label printer |