1. Visualization of tissue and plasma kallikreins and kinin B1 and B2 receptors in human lung carcinomas and mesothelioma
Jessica Chee

2. Hypothesis
Tissue (hK1) and plasma (hKB1) kallikreins and kinin B1 and B2 receptors are expressed in primary squamous carcinoma, adenocarcinoma, large cell carcinoma, small cell carcinoma, and carcinoid tumours of the lung and mesothelioma.

3. Purpose of the Study
Understanding the expression and cellular distribution of the kallikrein-kinin cascade proteins in different cancer cells would be important in determining whether the kinin dimeric antagonists could be of therapeutic value in the different lung cancer subtypes.

4. Background Epidemiology of lung carcinoma: Pathology:
● leading cause of cancer death in men (Franklin, 2000)
● Strong causal relationship between cigarette smoking and deaths from lung cancer (Doll et.al. 2004)
Pathology:
● A multi-step process, associated with multifocal metaplasia or dysplasia in the airways – field carcinogenesis
Classification: 2 major groups
Carcinoid- best prognosis
Small cell- usually have already spread by the time of presentation, surgery unlikely to be curative
Small cell carcinoma
● Small cell carcinoma
● Carcinoid
Non-small cell carcinoma
● adenocarcinoma
● squamous carcinoma
● large cell carcinoma

5. Kallikrein-kinin cascade proteins
TK (hK1) is implicated in carcinogenesis through the induction of gene expression (Dlamini et al 1999, Clements et al 2001)
Tissue (hK1) and plasma (hKB1) kallikreins - serine proteases that release bradykinin and lys-bradykinin from kininogens (Bhoola 1992)
2. Kinins are vasoactive peptides

6. ● Kinins act through B1 and B2 receptors, which are seven transmembrane receptors coupled to the heterotrimeric G proteins-
Gaq, Gγβ, Gas, and G12,13
●Kinins are mitogenic and enhance the proliferation of tumour cells (Robert and Gullick 1989)
● Kinins enhance metastasis through their vascular actions (Bhoola 2001, Wu et. al. 2002)

7. Increased permeability
Tumour growth
Pain
MMP activation
Tumour cells
Bradykinin NO
Prostaglandins
Metastasis
Vascular effects
angiogenesis
Vasodilatation
Increased permeability
Fluid accumulation
B2 receptor
Bradykinin and tumour metastasis
NO reacts with superoxide, which then activates fibroblast and neutrophil derived pro-MMPs  degradation of matrix proteins, increased permeability and extravasation

8. Aim
To investigate the expression and localization of tissue and plasma kallikreins and kinin B1 and B2 receptors in primary squamous carcinoma, adenocarcinoma, large cell carcinoma, small cell carcinoma, and carcinoid tumours of the lung and mesothelioma.

9. Method
● Tissue sections were kindly provided by Dr Anup Naran, PathCentre WA.
● No. of samples- 3 per each carcinoma, 20 sequential sections per each tumour sample.
Procedure:
● Dewaxed and rehydrated
● Endogenous peroxidase quenched
● Antigen retrieval
● Non specific binding blocked using: DAKO biotin block, DAKO protein block, 10% human serum, and 20% swine serum
● Primary antibody – rabbit anti-human B1, B2, PK (hKB) or TproK (hK1) antibodies
● Biotinylated secondary antibody
● Streptavidin-HRP
● Chromogens- DAB and AEC combined
● Mounted with Depex
Slides containing paraffin embedded tumour sections were obtained from the Dept. of Tissue Pathology, PathCentre, WA.
The sections were labelled with specific antibodies against B1 and B2 receptors and plasma- and tissue-kallikrein.
This was then visualized using the immunoperoxidase method and viewed by light microscopy

10. Negative controls
● Primary antibody omitted
● Non-immune rabbit serum
● Preabsorption of primary antibody
● Specificity test: antibody dilution
Positive controls
● Normal salivary gland
● Renal tubules
● Renal clear cell carcinoma

11. TproKallikrein
Plasma kallikrein
B1 receptor
B2 receptor

12. Optimization- Antibody dilutions
TK 1:50
TK 1:100
TK 1:200
TK 1:400

13. H & E stains
Adeno Squamous Large cell
Small cell Carcinoid Meso

14. Negative controls UNC Saliv squamous meso adeno
Omit Saliv squamous meso adeno

15. Tissue-proKallikrein (hK1)
Adenocarcinoma
Squamous carcinoma
Large cell carcinoma
Small cell carcinoma
Carcinoid tumour
Mesothelioma

16. Plasma kallikrein (hKB)
Adenocarcinoma
Squamous carcinoma
Large cell carcinoma
Small cell carcinoma
Carcinoid tumour
Mesothelioma

17. Kinin B1 receptor Adenocarcinoma Squamous carcinoma
Large cell carcinoma
Small cell carcinoma
Carcinoid tumour
Mesothelioma

18. Kinin B2 receptor Adenocarcinoma Squamous carcinoma
Large cell carcinoma
Small cell carcinoma
Carcinoid tumour
Mesothelioma

19. Looking ahead Work to be done:
● The labelling will be quantified using image analysis and values expressed as pixels/μm2
● Future experiments:
● N value for each to be increased to 6
● In situ hybridisation / Real time PCR
● Expression of additional kallikriens in lung carcinomas

20. A special thank you to: Conny, Rob, Sally Dr Anup Naran Dr Neil Misso
Prof Kanti Bhoola
Prof Phil Thompson